Journal: Nature Cell Biology

Article Title: Aggresome assembly at the centrosome is driven by CP110–CEP97–CEP290 and centriolar satellites

doi: 10.1038/s41556-022-00869-0

Figure Lengend Snippet: ( a ) RPE-1 cells were transiently transfected with GFP-HTT97Q and stained for pHSP27 or Ub + . DNA was stained with DAPI. Scale bar 10 μm. ( b ) Immunoblot of soluble and insoluble fractions from WT and PCM1 KO cells transfected with GFP-HTT25Q. GAPDH and Histone H3 were used controls for the soluble and insoluble fractions, respectively. ( c ) Cells were transfected with GFP-HTT97Q for 5 hours, then treated with DMSO or nocodazole for 5 hours before being fixed and stained for GFP and α-tubulin. Scale bar 10 μm. ( d ) Immunoblot of soluble and insoluble fractions from cells prepared as in c . GAPDH and Histone H3 were used controls for the soluble and insoluble fractions, respectively. ( e ) WT and STIL KO U-2 OS cells were transiently transfected with GFP-HTT97Q then stained for GFP, CEP135 and α-tubulin. Scale bar 10 μm. ( f ) Immunoblot of soluble and insoluble fractions from cells prepared as in e . GAPDH and Histone H3 were used controls for the soluble and insoluble fractions, respectively. ( g ) HEK293T-FLAG-miniTurbo-CP110 cells were treated and stained as indicated. Scale bar 10 μm, 2 μm. ( h ) Immunoblot of HEK293T-FLAG-miniTurbo-CP110 cells treated and probed as indicated. α-tubulin was used as a loading control. ( i ) The number of preys from DMSO- or MG132-treated groups. Preys were defined as detailed in the Methods. ( j ) Functional enrichment analysis was performed with the preys from i . using g:Profiler and the KEGG database. ( k ) Spectral counts from the genes which are implicated in Parkinson’s disease, proteasome function or Huntington’s disease are shown. Genes which are related to the respective pathways are marked in green. Unprocessed immunoblots are provided as source data. For i , j , k : the raw mass spectrometry data and analysis giving rise to these panels is available in Supplementary Table .

Article Snippet: To produce lentivirus, 4 × 10 6 HEK293T (female, human kidney; ATCC, ACS-4500) cells were seeded in a T-75 flask and transfected with the relevant lentiviral transfer vector, 3 μg psPAX2 and 2 μg pCMV-VSV-G using Lipofectamine 3000 (Invitrogen).

Techniques: Transfection, Staining, Western Blot, Control, Functional Assay, Mass Spectrometry

Journal:

Article Title: Biochemical Characterization of Human Epidermal Retinol Dehydrogenase 2

doi: 10.1016/j.cbi.2008.09.019

Figure Lengend Snippet: HEK293 cells were transfected with either empty vector (A) or RDH-E2 expression vector (B) and incubated with anti-FLAG antibodies. RDH-E2 expression was visualized using FITC-conjugated antibodies. Endoplasmic reticulum of HEK293 cells was visualized by staining with FITC-conjugated concanavalin A (C).

Article Snippet: Human embryonic kidney 293 (HEK293) cells (American Type Culture Collection, Manassas, VA) were cultured in minimal essential medium containing 10% horse serum and penicillin/streptomycin at 37 °C with 5% CO 2 .

Techniques: Transfection, Plasmid Preparation, Expressing, Incubation, Staining

Journal:

Article Title: Biochemical Characterization of Human Epidermal Retinol Dehydrogenase 2

doi: 10.1016/j.cbi.2008.09.019

Figure Lengend Snippet: HEK293 cells transfected with expression vector for RDH-E2 (+) or empty vector (−) were incubated with tritiated androsterone (ADT), dihydrotestosterone (DHT), allopregnanolone (ALLO), dehydroepiandrosterone (DHEA); progesterone (PROG), or corticosterone (CORT).

Article Snippet: Human embryonic kidney 293 (HEK293) cells (American Type Culture Collection, Manassas, VA) were cultured in minimal essential medium containing 10% horse serum and penicillin/streptomycin at 37 °C with 5% CO 2 .

Techniques: Transfection, Expressing, Plasmid Preparation, Incubation

Journal: medRxiv

Article Title: Within-host evolution of SARS-CoV-2 in an immunosuppressed COVID-19 patient: a source of immune escape variants

doi: 10.1101/2021.04.30.21256244

Figure Lengend Snippet: ( a ) Schematic overview of the sequence variations in SARS-CoV-2 genomes detected in early (d14) or late (d105) swab samples and isolated viruses. The heatmap illustrates the positions and the frequency of major variations in the viral genome (cut off 15%). The days of isolation are indicated at the right. The heatmap colors represent the variant frequencies. In ORF7b, L14* indicates a frame shift mutation due to a deletion of two nucleotides. ( b ) Immunofluorescence analysis of SARS-CoV-2 infected cell cultures. VeroE6 cells were infected with one of the two virus isolates (d14 or d105) using a multiplicity of infection (moi) of 0.1 plaque forming units (pfu)/cell. At 8 h post infection, the cells were fixed and stained with SARS-CoV-2 N- and S-specific antibodies (red). In addition, F-actin (white) and nuclear DNA (DAPI, blue) were detected. ( c ) Western blot analysis of viral protein expression. Calu-3 cells were infected with a moi of 0.001 pfu/cell with either of the two virus isolates (d14 or d105). Cells were lysed 8h, 24h, 48h and 72h post-infection and analyzed using N- and S-specific antibodies. Detection of β-actin was used as loading control. (Fig.4 continued on the following page) ( d ) Growth of the two patient isolates in VeroE6 and Calu-3 cells. The cells were infected with either of the two patient isolates (d14 or d105) using a moi of 0.001 pfu/cell. At different time points post infection, cell culture supernatants were collected and viral titers were determined by plaque assay on VeroE6 cells. The log-transformed titers are shown as means ± SD of results from three independent experiments. Significance was determined via two-way ANOVA with a Sidak’s multiple comparison test, **p<0.0021, ***p<0.0002, ns=non significant. (E and F) Late SARS-CoV-2 isolate is attenuated in mice. Weight loss ( e ) and survival ( f ) of 8 to 12 weeks-old K18-hACE2 mice intranasally infected with 200 or 2000 pfu of d14 or d105 viruses. Signs of disease and body weight loss were monitored daily for 14 days. The weight loss is visualized as mean ± SEM. Significance was determined via two-way ANOVA with a Sidak’s multiple comparison test, *p<0.0332, **p<0.0021, ***p<0.0002.

Article Snippet: Filtered throat swab samples were inoculated on African green monkey kidney VeroE6 cells (ATCC CRL-1586) (2 x 10 cells) in 4 ml DMEM with 2% FCS and incubated at 37°C and 5% CO2 for 4 to 6 days until cytopathic effect was visible.

Techniques: Sequencing, Isolation, Variant Assay, Mutagenesis, Immunofluorescence, Infection, Virus, Staining, Western Blot, Expressing, Control, Cell Culture, Plaque Assay, Transformation Assay, Comparison

Journal: medRxiv

Article Title: Within-host evolution of SARS-CoV-2 in an immunosuppressed COVID-19 patient: a source of immune escape variants

doi: 10.1101/2021.04.30.21256244

Figure Lengend Snippet: ( a and b ) Detection of neutralizing activity of immune sera against SARS-CoV-2 variants. 100 pfu of the d14 and d105 isolates were incubated for 60 min at room temperature with serial dilutions of the patient sera. Sera obtained from naïve (– ctrl) or convalescent individuals (+ ctrl) served as negative and positive controls. Virus neutralization was determined by plaque assay on VeroE6 cells. Virus titers are indicated as percentages (mean ±SD) of the titer of the untreated virus inoculum. The dotted lines indicate the cutoff value between positive (<50%) and negative (>50%) neutralization. ( a ) Sera from the immunocompromised patient. The times of blood withdrawal are indicated. ( b ) Convalescent sera from COVID-19 patients suffering from mild, moderate or severe disease or human post vaccination (BNT162b2 mRNA) sera. ( c-e ) Neutralization capacity of SARS-CoV-2 antisera using VSV*ΔG(FLuc) vector pseudotyped with the SARS-CoV-2 spike protein and coding for firefly luciferase. The pseudotyped viruses were incubated with serial dilutions of a COVID-19 convalescent serum prior to inoculation of VeroE6 cells. Pseudotyped virus infection was monitored 16 h post infection by measuring the firefly luciferase activity in the cell lysates. ( c ) Neutralization of VSV*ΔG(FLuc) pseudotyped with the early and late SARS-CoV-2 spike variants (d14 and d105) using serial dilutions of a COVID-19 convalescent serum. ( d and e ) Neutralization of VSV*ΔG(FLuc) pseudotyped with the d14 spike protein containing the individual or combined mutations characteristically found in the late d105 and d140 variants. Immune serum from a convalescent COVID-19 patient ( d ) or a vaccinated person ( e ) were analyzed. The neutralization was determined by calculating the NT 50 via a non-linear regression (variable slope, four parameters). Shown are means ± SD (n=3). Statistics were calculated with a one-way ANOVA (Tukey’s multiple comparison test), ns = non-significant, *p<0.0332, **p<0.0021, ***p<0.0002.

Article Snippet: Filtered throat swab samples were inoculated on African green monkey kidney VeroE6 cells (ATCC CRL-1586) (2 x 10 cells) in 4 ml DMEM with 2% FCS and incubated at 37°C and 5% CO2 for 4 to 6 days until cytopathic effect was visible.

Techniques: Activity Assay, Incubation, Virus, Neutralization, Plaque Assay, Plasmid Preparation, Luciferase, Infection, Comparison

Journal: medRxiv

Article Title: Within-host evolution of SARS-CoV-2 in an immunosuppressed COVID-19 patient: a source of immune escape variants

doi: 10.1101/2021.04.30.21256244

Figure Lengend Snippet: Sera were collected from convalescent K18-hACE2 mice at least 21 days post infection with Muc-IMB-1 (n=5), d14 (n=2) or d105 (n=13) virus isolates. ( a ) Anti-SARS-CoV-2 IgG titers of serially diluted sera (mean ±SD) were determined using virus-infected cells and indirect immunofluorescence analysis (IFA). ( b and c ) Neutralization of d14 and d105 virus isolates by convalescent mouse sera obtained after infection with wild-type SARS-CoV-2, d14 and Muc-IMB-1 (anti-wt sera, n=7) ( b ), or with variant d105 virus isolate (anti-d105 sera, n=13) ( c ). Neutralization capacity was determined by incubating 100 pfu of either virus isolate with serial dilutions of the mouse sera. The mixture was then applied to VeroE6 cells and infected cells were stained with N-specific antibodies. The highest dilution for each individual serum causing 50% neutralization was plotted as neutralizing titer. ( d and e ) Neutralization of B.1.1.7 and B.1.351 variants of concern by mouse convalescent sera was determined as described in panels b and c. 100 pfu of the variant viruses were incubated with dilutions of sera from mice infected with wild-type SARS-CoV-2 (wt) ( d ) or d105 isolate ( e ). Neutralization titers are meant as the highest dilution for each individual serum causing 50% reduction of infectivity. Each serum titer (b - e) is shown as mean out of two independent experiments. Significance was determined via a paired t test with *p<0.0332, **p<0.0021, ***p<0.0002. ( f and g ) Convalescent animals are protected against re-challenge infection. Weight loss ( f ) and survival ( g ) of convalescent K18-hACE2 mice (mean ± SEM), challenged one to four months after the prime infection. Animals primarily infected with d14 and Muc-IMB-1 viruses (pooled wt survivors, n=7), or with d105 virus (n=14) were intranasally challenged with 100,000 pfu of d14 or d105 viruses (2 to 7 mice per group, as indicated). As a control, naïve 8 weeks old K18-hACE2 mice were intranasally infected with 100,000 pfu of d14 or d105 isolate viruses (n=2 per group).

Article Snippet: Filtered throat swab samples were inoculated on African green monkey kidney VeroE6 cells (ATCC CRL-1586) (2 x 10 cells) in 4 ml DMEM with 2% FCS and incubated at 37°C and 5% CO2 for 4 to 6 days until cytopathic effect was visible.

Techniques: Infection, Virus, Immunofluorescence, Neutralization, Variant Assay, Staining, Incubation, Control

Journal:

Article Title: LPP, an Actin Cytoskeleton Protein Related to Zyxin, Harbors a Nuclear Export Signal and Transcriptional Activation Capacity

doi:

Figure Lengend Snippet: Characterization of LPP/VASP binding. (A) Preparation of VASP-enriched extracts. Total Vero cell extracts (lanes 1) or proteins bound to profilin-Sepharose after incubation with Vero cell extracts (lanes 2) were analyzed by SDS-PAGE and stained with Coomassie blue (left panel) or transferred onto a nitrocellulose membrane and stained with anti-VASP antibodies (right panel). The positions of molecular mass markers (kilodaltons) are shown on the left. (B) The N-terminal portion of LPP interacts with VASP in vitro. The profilin-bound proteins were separated by SDS-PAGE and transferred onto a nitrocellulose membrane. (Lanes 1–3) Identical membranes were incubated with myc-tagged GST-LPP3–414 (lane 1), with myc-tagged GST-LPP3–414 in the presence of a 100-fold molar excess of GST (lane 2), or without protein (lane 3) and then probed with anti-myc antibodies. (Lane 4) An identical membrane was probed directly with anti-VASP antibodies to indicate the positions of VASP bands. (Lane 5) A membrane was incubated with GST-ActA235–584 and then probed with anti-ActA3 (Golsteyn et al., 1997a ). The positions of molecular mass markers (kilodaltons) are shown on the left. (C) VASP coimmunoprecipitates with LPP. Vero cell extracts were incubated with nonimmune (lane 1) or purified MP2 (lane 2) antibody. Immunoprecipitates were analyzed by SDS-PAGE and Western blotting with mouse anti-VASP antibody. Total cell extracts were used to indicate the positions of VASP bands (lane 3). The positions of molecular mass markers (kilodaltons) are shown on the left. (D) Scheme of GFP-LPP chimera. GFP-LPP3–414-M is the fusion of Green Fluorescent Protein (GFP), the N-terminal part of LPP (amino acids 3–414), the myc tag, and the membrane anchor of ActA protein (amino acids 613–639). This ActA sequence is able to target proteins to the outer membrane of mitochondria. (E) Production of GFP-LPP3–414-M chimera in transiently transfected HeLa cells. Total extracts of nontransfected (lane 1) or transiently transfected HeLa cells producing GFP-LPP3–414-M (lane 2) were analyzed by SDS-PAGE and Western blotting with LPP2 antiserum. The antibody recognizes a band at 90 kDa that is not present in nontransfected cells. The arrow indicates the position of endogenous LPP. The positions of molecular mass markers (kilodaltons) are shown on the left. (F) LPP recruits VASP to an ectopic mitochondrial localization. HeLa cells transiently transfected to produce GFP-LPP3–414-M were fixed, permeabilized, and stained with anti-VASP antibody. Note that in addition to structures identified as mitochondria in separate experiments, VASP also localizes to focal adhesions, as in a nontransfected cell (inset). Bar, 20 μm.

Article Snippet: Cell Lines and Cells The human cervix carcinoma HeLa cell line (American Type Culture Collection [ATCC; Rockville, MD] CCL-2), African green monkey kidney Vero cell line (ATCC CCL-81), porcine proximal kidney epithelial LLC-PK1 cell line (ATCC CL-101), human colon adenocarcinoma CaCo2 cell line (ATCC HTB-37), human penis carcinoma keratinocytes ( Rogel-Gaillard et al. , 1992 ; Robine et al. , 1993 ), and human foreskin fibroblasts (HFF) (Dr. A. Rochat, Ecole Normale Superieure, Paris, France) were grown in DMEM supplemented with 10% FCS (complete medium).

Techniques: Binding Assay, Incubation, SDS Page, Staining, Membrane, In Vitro, Purification, Western Blot, Sequencing, Transfection

Journal: Journal of Virology

Article Title: Entry of Human Coronavirus NL63 into the Cell

doi: 10.1128/JVI.01933-17

Figure Lengend Snippet: Importance of endosomal entry for HCoV-NL63 infection. (A) Inhibition of HCoV-NL63 infection in LLC-Mk2 cells and HAE cultures by the lysosomotropic agents ammonium chloride (NH4Cl) (50 mM) and bafilomycin A (Baf A) (100 nM), as determined by RT-qPCR. Data on the y axis represent LRVs. The assay was performed in triplicate, and average values with standard errors are presented. P values of <0.05 were considered significant and are denoted with an asterisk. (B) The cytotoxicity of the tested inhibitors was measured with an XTT assay. Data on the y axis represent viability of the treated cells compared to the untreated reference samples. The assay was performed in triplicate, and average values with standard errors are presented. (C and D) Confocal images showing colocalization of HCoV-NL63 virions with the early endosomal marker EEA1 on LLC-Mk2 cells (C) and HAE cultures (D). Scale bars = 5 μm. Green, HCoV-NL63; red, EEA1.

Article Snippet: LLC-Mk2 cells (ATCC CCL-7; Macaca mulatta kidney epithelial) were maintained in minimal essential medium (MEM) (two parts Hanks' MEM and one part Earle's MEM; Thermo Fisher Scientific, Poland) supplemented with 3% heat-inactivated fetal bovine serum (Thermo Fisher Scientific, Poland), penicillin (100 U/ml), streptomycin (100 μg/ml), and ciprofloxacin (5 μg/ml).

Techniques: Infection, Inhibition, Quantitative RT-PCR, XTT Assay, Marker

Journal: Journal of Virology

Article Title: Entry of Human Coronavirus NL63 into the Cell

doi: 10.1128/JVI.01933-17

Figure Lengend Snippet: HCoV-NL63 binding to ACE2 triggers clathrin-mediated endocytosis. Precooled LLC-Mk2 cells were incubated with gradient-purified HCoV-NL63 for 40 min at 4°C following 0 min (A and B) or 5 min (C) of incubation at 32°C. Colocalization of the virus (green) and ACE2 (red) was analyzed using confocal microscopy (A). No colocalization with clathrin was observed after 0 min of incubation (B). Triple colocalization of virus with ACE2 and clathrin (blue) is visible in panel C. Images on the right side are zoomed-in regions indicated by white rectangles on the left-side slides. A representative image is shown. Scale bars = 10 μm.

Article Snippet: LLC-Mk2 cells (ATCC CCL-7; Macaca mulatta kidney epithelial) were maintained in minimal essential medium (MEM) (two parts Hanks' MEM and one part Earle's MEM; Thermo Fisher Scientific, Poland) supplemented with 3% heat-inactivated fetal bovine serum (Thermo Fisher Scientific, Poland), penicillin (100 U/ml), streptomycin (100 μg/ml), and ciprofloxacin (5 μg/ml).

Techniques: Binding Assay, Incubation, Purification, Confocal Microscopy

Journal: Journal of Virology

Article Title: Entry of Human Coronavirus NL63 into the Cell

doi: 10.1128/JVI.01933-17

Figure Lengend Snippet: HCoV-NL63 colocalizes with clathrin but not caveolin. (A and B) LLC-Mk2 cells were incubated with gradient-purified HCoV-NL63 for 40 min at 4°C following 5 min (A) or 20 min (B) of incubation at 32°C. HAE cultures were incubated with gradient-purified HCoV-NL63 for 40 min at 4°C following 120 min of incubation at 32°C. HCoV-NL63 colocalization with clathrin (A) or caveolin (B) was analyzed with confocal microscopy (HCoV-NL63, green; clathrin and caveolin, red; nuclei, blue). (C) Cells mock incubated and stained with isotypic antibodies were used as a control. Scale bars = 5 μm.

Article Snippet: LLC-Mk2 cells (ATCC CCL-7; Macaca mulatta kidney epithelial) were maintained in minimal essential medium (MEM) (two parts Hanks' MEM and one part Earle's MEM; Thermo Fisher Scientific, Poland) supplemented with 3% heat-inactivated fetal bovine serum (Thermo Fisher Scientific, Poland), penicillin (100 U/ml), streptomycin (100 μg/ml), and ciprofloxacin (5 μg/ml).

Techniques: Incubation, Purification, Confocal Microscopy, Staining

Journal: Journal of Virology

Article Title: Entry of Human Coronavirus NL63 into the Cell

doi: 10.1128/JVI.01933-17

Figure Lengend Snippet: Clathrin and dynamin inhibitors hamper internalization of HCoV-NL63. (A to C) In order to verify the effectiveness of inhibitors, LLC-Mk2 cells were incubated with control DMSO (A), 10 μM Pitstop 2 (B), or 10 μM MiTMAB (C) for 30 min at 37°C and inoculated with Alexa Fluor 488-labeled transferrin. Following incubation (45 min, 37°C), cells were fixed and stained for actin (red). Transferrin entry was evaluated with confocal microscopy. (E to G) LLC-Mk2 cells were incubated with control DMSO (E), 10 μM Pitstop 2 (F), or 10 μM MiTMAB (G) for 30 min at 37°C. Cells were inoculated with purified HCoV-NL63 and incubated at 32°C for 1 h. Subsequently, cells were fixed and immunostained for HCoV-NL63 particles (green) and actin (red). (D) Mock-infected cells were used as a control. Scale bars = 10 μm.

Article Snippet: LLC-Mk2 cells (ATCC CCL-7; Macaca mulatta kidney epithelial) were maintained in minimal essential medium (MEM) (two parts Hanks' MEM and one part Earle's MEM; Thermo Fisher Scientific, Poland) supplemented with 3% heat-inactivated fetal bovine serum (Thermo Fisher Scientific, Poland), penicillin (100 U/ml), streptomycin (100 μg/ml), and ciprofloxacin (5 μg/ml).

Techniques: Incubation, Labeling, Staining, Confocal Microscopy, Purification, Infection

Journal: Journal of Virology

Article Title: Entry of Human Coronavirus NL63 into the Cell

doi: 10.1128/JVI.01933-17

Figure Lengend Snippet: Cytotoxicity of Pitstop 2 and MiTMAB on LLC-Mk2 cells. The cytotoxicity of the endocytosis inhibitors was tested with an XTT assay. Cells were incubated with control DMSO, 10 μM Pitstop 2, or 10 μM MiTMAB for 2 h at 37°C Data on the y axis represent viability of the treated cells compared to the untreated reference samples. The assay was performed in triplicate, and average values with standard errors are presented.

Article Snippet: LLC-Mk2 cells (ATCC CCL-7; Macaca mulatta kidney epithelial) were maintained in minimal essential medium (MEM) (two parts Hanks' MEM and one part Earle's MEM; Thermo Fisher Scientific, Poland) supplemented with 3% heat-inactivated fetal bovine serum (Thermo Fisher Scientific, Poland), penicillin (100 U/ml), streptomycin (100 μg/ml), and ciprofloxacin (5 μg/ml).

Techniques: XTT Assay, Incubation

Journal: Journal of Virology

Article Title: Entry of Human Coronavirus NL63 into the Cell

doi: 10.1128/JVI.01933-17

Figure Lengend Snippet: Numerical image analysis: clathrin and dynamin inhibitors block HCoV-NL63 entry. (B to D) LLC-Mk2 cells were incubated with DMSO (B), 10 μM MiTMAB (C), or 10 μM Pitstop 2 (D) for 30 min at 37°C and subsequently inoculated with purified HCoV-NL63 and incubated for 45 min at 32°C. Confocal images were digitalized, and the localization of each virus particle relative to the cellular membrane was assessed. (A) Number of internalized virus particles relative to number of virions on the cell surface (y axis) for cells treated with DMSO (control), Pitstop 2, or MiTMAB. In panels B, C, and D, raw data for cells treated with DMSO, Pitstop 2, or MiTMAB, respectively, are presented. Histograms show the average number of virus particles (y axis) versus the distance from the cell surface (x axis). Values of <0 on the x axis indicate that the virus is inside the cell, while for extracellular virions, the x value is ≥0.

Article Snippet: LLC-Mk2 cells (ATCC CCL-7; Macaca mulatta kidney epithelial) were maintained in minimal essential medium (MEM) (two parts Hanks' MEM and one part Earle's MEM; Thermo Fisher Scientific, Poland) supplemented with 3% heat-inactivated fetal bovine serum (Thermo Fisher Scientific, Poland), penicillin (100 U/ml), streptomycin (100 μg/ml), and ciprofloxacin (5 μg/ml).

Techniques: Blocking Assay, Incubation, Purification

Journal: Journal of Virology

Article Title: Entry of Human Coronavirus NL63 into the Cell

doi: 10.1128/JVI.01933-17

Figure Lengend Snippet: Clathrin and dynamin inhibitors limit the number of LLC-Mk2 infected cells. (A to D) LLC-Mk2 cells were incubated with control DMSO (A), 5 μg/ml nystatin (B), 10 μM MiTMAB (C), or 10 μM Pitstop 2 (D) for 1 h at 37°C and inoculated with HCoV-NL63 (TCID50 = 100/ml). After 2 h of incubation at 32°C, virions that were not internalized were inactivated with acidic buffer (pH 3), and cells were incubated for 4 days at 32°C in the presence of the tested inhibitors or control DMSO. (E and F) The identical procedure was applied to cells, but in these MiTMAB (E) and Pitstop 2 (F) were applied after the acid wash. Fixed cells were immunostained with anti-NL63 nucleocapsid protein (green) and nuclei (blue), and confocal images were collected. Scale bar = 200 μm.

Article Snippet: LLC-Mk2 cells (ATCC CCL-7; Macaca mulatta kidney epithelial) were maintained in minimal essential medium (MEM) (two parts Hanks' MEM and one part Earle's MEM; Thermo Fisher Scientific, Poland) supplemented with 3% heat-inactivated fetal bovine serum (Thermo Fisher Scientific, Poland), penicillin (100 U/ml), streptomycin (100 μg/ml), and ciprofloxacin (5 μg/ml).

Techniques: Infection, Incubation

Journal: Journal of Virology

Article Title: Entry of Human Coronavirus NL63 into the Cell

doi: 10.1128/JVI.01933-17

Figure Lengend Snippet: Clathrin and dynamin inhibitors hamper replication of HCoV-NL63 in LLC-MK2 cells. (A) HCoV-NL63 replication in LLC-Mk2 cells and HAE cultures in the presence of entry inhibitors or control DMSO was analyzed using RT-qPCR. Cultures were incubated with 10 μM Pitstop 2, 10 μM MiTMAB, 5 μg/ml nystatin, or DMSO for 1 h at 37°C and inoculated with HCoV-NL63 (TCID50 = 400/ml). After 2 h of incubation at 32°C, virions that were not internalized were inactivated with acidic buffer (pH 3), and cells were incubated for 5 days at 32°C. The data are presented as log reduction value (LRV) compared to the control sample. The assay was performed in triplicate, and average values with standard errors are presented. P values of <0.05 were considered significant and are denoted with an asterisk. (B) The cytotoxicity of the inhibitors was tested with an XTT assay. Cells were incubated with 10 μM Pitstop 2, 10 μM MiTMAB, 5 μg/ml nystatin, or DMSO for 5 days at 32°C. Data on the y axis represent viability of the treated cells compared to the untreated reference samples. The assay was performed in triplicate, and average values with standard errors are presented.

Article Snippet: LLC-Mk2 cells (ATCC CCL-7; Macaca mulatta kidney epithelial) were maintained in minimal essential medium (MEM) (two parts Hanks' MEM and one part Earle's MEM; Thermo Fisher Scientific, Poland) supplemented with 3% heat-inactivated fetal bovine serum (Thermo Fisher Scientific, Poland), penicillin (100 U/ml), streptomycin (100 μg/ml), and ciprofloxacin (5 μg/ml).

Techniques: Quantitative RT-PCR, Incubation, XTT Assay

Journal: Journal of Virology

Article Title: Entry of Human Coronavirus NL63 into the Cell

doi: 10.1128/JVI.01933-17

Figure Lengend Snippet: TMPRSS2 is required for entry into HAE cells but does not enable virus-cell fusion on the cell surface. (A) HCoV-NL63 replication in LLC-Mk2 cells and HAE cultures in the presence of camostat or control DMSO was analyzed using RT-qPCR. Cultures were incubated with 100 μM camostat or DMSO for 1 h at 37°C and inoculated with HCoV-NL63 (TCID50 = 400/ml). After 2 h of incubation at 32°C, virions that were not internalized were inactivated with acidic buffer (pH 3), and cells were incubated for 5 days at 32°C. The data are presented as log reduction value (LRV) compared to the control sample. The assay was performed in triplicate, and average values with standard errors are presented. P values of <0.05 were considered significant and are denoted with asterisks. (B) HAE cultures were incubated with control DMSO or 100 μM camostat for 1 h at 37°C. Further, cells were inoculated with purified HCoV-NL63 and incubated at 32°C for 2 h. Subsequently, cells were fixed and immunostained for HCoV-NL63 particles (green), actin (red), and nuclei (blue). Scale bars = 5 μm.

Article Snippet: LLC-Mk2 cells (ATCC CCL-7; Macaca mulatta kidney epithelial) were maintained in minimal essential medium (MEM) (two parts Hanks' MEM and one part Earle's MEM; Thermo Fisher Scientific, Poland) supplemented with 3% heat-inactivated fetal bovine serum (Thermo Fisher Scientific, Poland), penicillin (100 U/ml), streptomycin (100 μg/ml), and ciprofloxacin (5 μg/ml).

Techniques: Quantitative RT-PCR, Incubation, Purification

Journal: Journal of Virology

Article Title: Entry of Human Coronavirus NL63 into the Cell

doi: 10.1128/JVI.01933-17

Figure Lengend Snippet: Actin is important for HCoV-NL63 entry. LLC-MK2 cells were incubated with DMSO (A), 10 μM cytochalasin D (B and E), 1.5 μM jasplakinolide (C and F), or 400 nM nocodazole (D and G) for 1 h at 37°C and then inoculated with purified HCoV-NL63 and incubated at 32°C for 1 h. Actin and virus localization was verified with confocal microscopy; fixed cells were immunostained for HCoV-NL63 particles (green), actin (red), and nuclei (blue). Scale bars = 10 μm.

Article Snippet: LLC-Mk2 cells (ATCC CCL-7; Macaca mulatta kidney epithelial) were maintained in minimal essential medium (MEM) (two parts Hanks' MEM and one part Earle's MEM; Thermo Fisher Scientific, Poland) supplemented with 3% heat-inactivated fetal bovine serum (Thermo Fisher Scientific, Poland), penicillin (100 U/ml), streptomycin (100 μg/ml), and ciprofloxacin (5 μg/ml).

Techniques: Incubation, Purification, Confocal Microscopy

Journal: Journal of Virology

Article Title: The 5′ Untranslated Region of Human Bocavirus Capsid Transcripts Regulates Viral mRNA Biogenesis and Alternative Translation

doi: 10.1128/JVI.00443-18

Figure Lengend Snippet: 5′ UTR regulated HBoV1 capsid mRNA abundance and protein translation. (A) Diagram of HBoV1 capsid expression constructs with the T7 promoter for the in vitro assay. (B) In vitro coupled transcription/translation assay. In vitro assays were performed according to the manufacturer's instructions. Expressed proteins were run on a 15% SDS-PAGE gel, and the signal was detected with a Cyclone Plus system (PerkinElmer) and analyzed using OptiQuant software. The ratio of VP1 to VP3 is presented at the bottom of the gel. The experiment was repeated at least three times. (C) Diagram of HBoV1 VP cDNA constructs with the cytomegalovirus (CMV) promoter. (D) Northern blot. Ten micrograms of total RNAs prepared from transfected cells were resolved on 1.5% agarose gels, transferred to Hybond-N + membranes, and hybridized with probes spanning nt 349 to 5167. The signal was detected using a ChemiDoc MP imaging system (Bio-Rad). Ethidium bromide (EB)-stained 18S RNA bands are shown as the loading control. (E) Western blot (WB). The lysates of HEK293T cells transfected with the plasmids described in panel C were analyzed using an anti-Flag antibody to detect capsid expression. β-Actin served as the loading control.

Article Snippet: The human embryonic kidney cell line HEK293T (ATCC, CRL-11268) was maintained in Dulbecco's modified Eagle medium (DMEM; Invitrogen) supplemented with 10% fetal bovine serum at 37°C in a humidified incubator with 5% CO 2 .

Techniques: Expressing, Construct, In Vitro, SDS Page, Software, Northern Blot, Transfection, Imaging, Staining, Control, Western Blot

Journal: Journal of Virology

Article Title: The 5′ Untranslated Region of Human Bocavirus Capsid Transcripts Regulates Viral mRNA Biogenesis and Alternative Translation

doi: 10.1128/JVI.00443-18

Figure Lengend Snippet: Identification of the cis elements in the 5′ UTR that regulated RNA abundance and translation. (A) Diagram of green fluorescent protein (GFP) reporter plasmids. Group I plasmids contained either the entire 5′ UTR or VP1 unique sequences. Group II plasmids contained both the 5′ UTR and VP1u. Group III constructs harbored VP1u with deletions in exons 1 to 4. Exon 4 in the plasmids of group IV was deleted. SV40, simian virus 40. (B) Microscopy of the fluorescent signal when reporter plasmids were transfected. (C) Western blot. The lysates of transfected HEK293T cells were analyzed using anti-GFP antibody, and β-actin served as the loading control. (D) Northern blot. Total RNAs were harvested from transfected cells, and Northern blotting was carried out as described in the legend to . Ethidium bromide (EB)-stained 18S RNA bands are shown as the loading control. The image in panel D was spliced from two different gels because the samples could not be run in one gel.

Article Snippet: The human embryonic kidney cell line HEK293T (ATCC, CRL-11268) was maintained in Dulbecco's modified Eagle medium (DMEM; Invitrogen) supplemented with 10% fetal bovine serum at 37°C in a humidified incubator with 5% CO 2 .

Techniques: Construct, Virus, Microscopy, Transfection, Western Blot, Control, Northern Blot, Staining

Journal: Journal of Virology

Article Title: The 5′ Untranslated Region of Human Bocavirus Capsid Transcripts Regulates Viral mRNA Biogenesis and Alternative Translation

doi: 10.1128/JVI.00443-18

Figure Lengend Snippet: 5′ UTR cis elements regulated HBoV1 capsid protein expression and mRNA abundance in HEK293T cells. (A) Diagram of capsid expression plasmids. Various lengths of the 5′ UTR sequence from p3XFlag-R6 were deleted to construct p3XFlag-Tru1, p3XFlag-Tru2, p3XFlag-E3 and -E4, and p3XFlag-mini. Plasmids p3XFlag-mini-T1 to p3XFlag-mini-T10 were generated by sequence deletion in exon 2 or exon 3 based on p3XFlag-mini, as indicated. (B) Western blot. The transfected HEK293T cell lysates were analyzed using anti-Flag antibody, and β-actin served as the loading control. (C) Northern blot. Total RNAs were harvested from transfected cells, and Northern blotting was carried out as described in the legend to . Ethidium bromide (EB)-stained 18S RNA bands are shown as the loading control.

Article Snippet: The human embryonic kidney cell line HEK293T (ATCC, CRL-11268) was maintained in Dulbecco's modified Eagle medium (DMEM; Invitrogen) supplemented with 10% fetal bovine serum at 37°C in a humidified incubator with 5% CO 2 .

Techniques: Expressing, Sequencing, Construct, Generated, Western Blot, Transfection, Control, Northern Blot, Staining

Journal: Journal of Virology

Article Title: The 5′ Untranslated Region of Human Bocavirus Capsid Transcripts Regulates Viral mRNA Biogenesis and Alternative Translation

doi: 10.1128/JVI.00443-18

Figure Lengend Snippet: The alternative translation of VP1 and VP2 was IRES independent. (A) Diagram of bicistronic fluorescent reporter constructs. The 3×Flag tag, the simian virus 40 poly(A) signal, and multiple-cloning sites (MCS) are denoted. (B) Microscopy images of fluorescent signals. Images of the green fluorescent protein (GFP) fluorescent signals are on the top, and images of the mCherry fluorescent signals are on the bottom (in images of the same visual fields). (C) Western blot. The lysates of transfected HEK293T cells were analyzed using anti-GFP and anti-Flag antibodies. β-Actin served as the loading control. (D) Northern blot. Total RNAs were harvested from transfected cells, and Northern blotting was carried out as described in the legend to . Ethidium bromide (EB)-stained 18S RNA bands are shown as the loading control.

Article Snippet: The human embryonic kidney cell line HEK293T (ATCC, CRL-11268) was maintained in Dulbecco's modified Eagle medium (DMEM; Invitrogen) supplemented with 10% fetal bovine serum at 37°C in a humidified incubator with 5% CO 2 .

Techniques: Construct, Virus, Cloning, Microscopy, Western Blot, Transfection, Control, Northern Blot, Staining

Journal: Journal of Virology

Article Title: The 5′ Untranslated Region of Human Bocavirus Capsid Transcripts Regulates Viral mRNA Biogenesis and Alternative Translation

doi: 10.1128/JVI.00443-18

Figure Lengend Snippet: The alternative translation of HBoV1 VP proteins was based on ribosomal leaky scanning. (A) Diagram of plasmids used for in vitro or in vivo translation. (B) Western blot. The lysates of transfected HEK293T cells were analyzed using anti-Flag antibody, and β-actin served as the loading control. (C) In vitro coupled transcription/translation assay. In vitro assays were performed as described in the legend to . (D) Northern blot. Total RNAs were harvested from transfected cells, and Northern blotting was carried out as described in the legend to . Ethidium bromide (EB)-stained 18S RNA bands are shown as the loading control.

Article Snippet: The human embryonic kidney cell line HEK293T (ATCC, CRL-11268) was maintained in Dulbecco's modified Eagle medium (DMEM; Invitrogen) supplemented with 10% fetal bovine serum at 37°C in a humidified incubator with 5% CO 2 .

Techniques: In Vitro, In Vivo, Western Blot, Transfection, Control, Northern Blot, Staining

Journal: Journal of Virology

Article Title: The 5′ Untranslated Region of Human Bocavirus Capsid Transcripts Regulates Viral mRNA Biogenesis and Alternative Translation

doi: 10.1128/JVI.00443-18

Figure Lengend Snippet: uATGs influenced HBoV1 capsid protein expression. (A) Diagram of HBoV1 uATG-mutated constructs. The positions of uATG1, uATG2, and uATG3 are denoted by their nucleotide sequence numbers. (B) Western blot. The transfected HEK293T cell lysates were analyzed using anti-Flag antibody, and β-actin served as the loading control. (C) Northern blot. Total RNAs were harvested from transfected cells, and Northern blotting was carried out as described in the legend to . Ethidium bromide (EB)-stained 18S RNA bands are shown as the loading control. ncRNA, noncoding RNA.

Article Snippet: The human embryonic kidney cell line HEK293T (ATCC, CRL-11268) was maintained in Dulbecco's modified Eagle medium (DMEM; Invitrogen) supplemented with 10% fetal bovine serum at 37°C in a humidified incubator with 5% CO 2 .

Techniques: Expressing, Construct, Sequencing, Western Blot, Transfection, Control, Northern Blot, Staining

Journal: Journal of Virology

Article Title: The 5′ Untranslated Region of Human Bocavirus Capsid Transcripts Regulates Viral mRNA Biogenesis and Alternative Translation

doi: 10.1128/JVI.00443-18

Figure Lengend Snippet: uATG2 and uATG3 knockout mutations in an HBoV1 infectious clone decreased viral progeny production. (A) Southern blot. Hirt DNA was isolated from HEK293T cells transfected with plasmids, as indicated, at 48 h posttransfection. The DpnI-digested fragments were resolved on 1% agarose gels and transferred to a Hybond-N + membrane, followed by hybridization with an HBoV1 probe spanning from nt 1 to 5543. dRF, double replication form; mRF, monomer replication form; ssDNA, single-stranded DNA. (B) Northern blot. Total RNAs were harvested from transfected cells, and Northern blotting was carried out as described in the legend to . Ethidium bromide (EB)-stained 18S RNA bands are shown as the loading control. The images in panel B were taken from different areas of the same photo or different gels and then joined together. (C) Quantitative PCR (qPCR) for the HBoV1 genome. Cell lysates of uATG-mutated HBoV1 infectious clones were prepared by four cycles of freezing and thawing, followed by DNase I digestion. The progeny viruses were separated by cesium chloride (CsCl) density gradient centrifugation, and genomic DNA was prepared as the template for qPCR. Means and standard deviations were calculated from the results of at least three independent experiments. The pHBoV plasmid was used as a control to establish a standard curve for absolute qPCR ( E = 105.0%, R 2 = 0.999, slope = −3.208). E, efficiency. (D) Western blot. uATG-mutated infectious clones were transfected into HEK293T cells, and Western blotting was performed with anti-NS, anti-NP, and anti-VP antibodies.

Article Snippet: The human embryonic kidney cell line HEK293T (ATCC, CRL-11268) was maintained in Dulbecco's modified Eagle medium (DMEM; Invitrogen) supplemented with 10% fetal bovine serum at 37°C in a humidified incubator with 5% CO 2 .

Techniques: Knock-Out, Southern Blot, Isolation, Transfection, Membrane, Hybridization, Northern Blot, Staining, Control, Real-time Polymerase Chain Reaction, Clone Assay, Gradient Centrifugation, Plasmid Preparation, Western Blot

Journal: Journal of Virology

Article Title: The 5′ Untranslated Region of Human Bocavirus Capsid Transcripts Regulates Viral mRNA Biogenesis and Alternative Translation

doi: 10.1128/JVI.00443-18

Figure Lengend Snippet: uATG mutations in an HBoV1 infectious clone altered viral RNA processing. RNase protection assay (RPA) with analysis of viral RNA polyadenylation at the (pA)p site. Ten micrograms of total RNAs prepared from HEK293T cells transfected with plasmids, as indicated, was protected by the (pA)p site-specific probe. RT, read through RNAs. The ratios of RNA polyadenylated at the (pA)p site versus read-through RNA are indicated as the mean and standard deviation. The numbers on the left are molecular size markers.

Article Snippet: The human embryonic kidney cell line HEK293T (ATCC, CRL-11268) was maintained in Dulbecco's modified Eagle medium (DMEM; Invitrogen) supplemented with 10% fetal bovine serum at 37°C in a humidified incubator with 5% CO 2 .

Techniques: Rnase Protection Assay, Transfection, Standard Deviation

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

Article Title: PD1 blockade enhances ICAM1-directed CAR T therapeutic efficacy in advanced thyroid cancer

doi: 10.1158/1078-0432.CCR-20-1523

Figure Lengend Snippet: (A) Representative flow cytometry plots of ATC cell lines 8505C and JV, and 293T and HeLa control cell lines, which are stained for ICAM1 (first row) and PD-L1 (second row) antibodies. FMO control and antibody-stained plots are indicated by unfilled and gray solid lines, respectively. Flow cytometry plots showing the level of expression of PD-L1 (gray solid line) and PD-L2 (red line) relative to FMO control (unfilled line) (third row). (B) Cytotoxicity of thyroid cancer patient-derived ICAM1-CAR T cells (for donor and CAR T information, refer to Supplementary Table 1) against target ATC cell lines and control cells were examined in combination with PD1 blocking antibody. Peripheral T cells were isolated from patients with ATC, PDTC, and WDPTC. 5×103 target cells were co-cultured either with non-transduced T (NT) cells or anti-ICAM1 CAR T cells at a 2.5:1 ratio (1.25×104 cells) in media (n = 3–7 per group and representative of two to three independent experiments). (C) Differences in target cell killing were compared at a timepoint when approximately 50% of the 8505C cells were killed (24 hr for ATC, 22 hr for PDTC, and 48 hr for WDPTC). Statistical significance was determined by one-way ANOVA for CAR T vs. CAR T + αPD1 and CAR T vs. non-transduced T (NT) cells. *, P < 0.05; **, P < 0.01; ***, P < 0.001. Data represents mean ± sem (results were confirmed by the Kruskal-Wallis test).

Article Snippet: The 8505C BRAF-mutated ATC cell line was obtained from DSMZ; HeLa cervical cancer cells and HEK 293T human embryonic kidney cells were obtained from ATCC.

Techniques: Flow Cytometry, Control, Staining, Expressing, Derivative Assay, Blocking Assay, Isolation, Cell Culture

Journal: Methods in Molecular Biology (Clifton, N.j.)

Article Title: Cell Adhesion Assays: Fabrication of an E-cadherin Substratum and Isolation of Lateral and Basal Membrane Patches

doi:

Figure Lengend Snippet: Characterization of basal membrane patches from MDCK cells on filters. (A,B) Staining with the membrane dye, DiIC18(3), of whole MDCK cells grown on filters (A), and isolated membrane patches (B), which show few internal organelles remaining after sonication. Bar, 10 μm. (C,D) Staining for the Golgi protein, p115 (red) and actin (green) on the basal membrane of intact MDCK cells grown on filters (C), and isolated basal membranes which retain actin fibers whereas p115-containing Golgi membranes are largely removed (D). (E,F) Staining for mitochondria (red) and actin filaments (green) of the basal membrane of intact MDCK cells (E), and isolated basal patches which show preservation of actin filaments but absence of mitochondria (F). Bar, 10 μm. Fixation of cells for Golgi and mitochondria staining was with 4% formaldehyde diluted in Ringers buffer. A polyclonal antibody to Golgi component p115 was a gift from Dr. Suzanne Pfeffer (Stanford University, Stanford, CA). A monoclonal antibody to mitochondrial Hsp70 was from Affinity Bioreagents. (G,H) GFP microtubules on the basal membrane of intact MDCK cells expressing GFP-tubulin (G), and isolated basal membranes, which show improved visibility of the cytoskeleton (H). Bar, 10 μm. green fluorescent protein -microtubules were fixed with 0.3% glutaraldehyde in BRB80 buffer, for 10 min for isolated membranes or 20 min for intact cells, with the addition of 0.1% Triton X-100 for intact cells.

Article Snippet: Madin-Darby canine kidney (MDCK) cells (American Type Culture Collection).

Techniques: Membrane, Staining, Isolation, Sonication, Preserving, Expressing

Journal: Methods in Molecular Biology (Clifton, N.j.)

Article Title: Cell Adhesion Assays: Fabrication of an E-cadherin Substratum and Isolation of Lateral and Basal Membrane Patches

doi:

Figure Lengend Snippet: Characterization of basal membrane patches from MDCK cells on E-cadherin:Fc substratum. (A) Staining with membrane dye, DiIC18(3), of isolated basal membrane patches on E-cadherin:Fc substratum. (B) Staining with monoclonal actin antibody (Chemicon). (C) Basal membranes on E-cadherin:Fc substratum stained with antibody directed against the intracellular domain of E-cadherin. (D) Staining with β-catenin antibody. All fixations were with 4% formaldehyde in Ringer’s buffer. Bar, 10 μm.

Article Snippet: Madin-Darby canine kidney (MDCK) cells (American Type Culture Collection).

Techniques: Membrane, Staining, Isolation

Journal: Methods in Molecular Biology (Clifton, N.j.)

Article Title: Cell Adhesion Assays: Fabrication of an E-cadherin Substratum and Isolation of Lateral and Basal Membrane Patches

doi:

Figure Lengend Snippet: Visualization of patches by differential interference contrast microscopy. MDCK clone II/G were plated on collagen-coated cover slips for 2 d, sonicated, and isolated membranes were imaged by differential interference contrast microscopy. Patches are outlined by asterisks. Note the actin fibers present on the membrane indicated by the arrow. Bar, 10 μm.

Article Snippet: Madin-Darby canine kidney (MDCK) cells (American Type Culture Collection).

Techniques: Microscopy, Sonication, Isolation, Membrane

Journal: Anesthesiology

Article Title: Critical Role of Interleukin-11 in Isoflurane-mediated Protection against Ischemic Acute Kidney Injury in Mice

doi: 10.1097/ALN.0b013e3182a950da

Figure Lengend Snippet: A and B. IL-11 mRNA measured with reverse transcription polymerase chain reaction (RTPCR) in HK-2 cells treated with 0-2.5% isoflurane for 6 h (A. N = 6) or 2.5% isoflurane for 0-6 h (B, N = 6). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA expression was quantified to normalize lane loading. Data are presented as means ± SD. * P < 0.05 vs. IL-11 mRNA measured after 0% isoflurane-treatment (A) or at 0 h (B). C. Isoflurane increases IL-11 protein (pg/ml) in cell culture media from HK-2 cells (N = 6). HK-2 cells were treated with 2.5% isoflurane or with carrier gas for 6 h or 16 h. * P < 0.05 vs. carrier gas treated group.

Article Snippet: Human and mouse proximal tubule cell culture and exposure to isoflurane Immortalized human renal proximal tubule (HK-2) cells (American Type Culture Collection, Manassas, VA) were grown and passaged with 50:50 mixture of Dulbecco’s Modified Eagle Media/F12 with 10% fetal bovine serum (Invitrogen, Carlsbad, CA) and antibiotics (100 U/ml penicillin G, 100 μg/ml streptomycin, and 0.25 μg/ml amphotericin B (Invitrogen) at 37°C in a 100% humidified atmosphere of 5% carbon dioxide-95% air.

Techniques: Reverse Transcription, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Expressing, Cell Culture

Journal: Anesthesiology

Article Title: Critical Role of Interleukin-11 in Isoflurane-mediated Protection against Ischemic Acute Kidney Injury in Mice

doi: 10.1097/ALN.0b013e3182a950da

Figure Lengend Snippet: A. IL-11 messenger ribonucleic acid (mRNA) (detected with reverse transcription polymerase chain reaction (RTPCR)) expression in HK-2 cells treated with 2.5% isoflurane for 6 h (N = 6). Representative images (top) and band intensity quantifications (bottom) expressed as fold increases in IL-11 expression over carrier gas plus immunoglobulin G (IgG) isotype antibody treated controls. B. IL-11 protein (detected with enzyme-linked immunosorbent assay (ELISA)) expression in HK-2 cells treated with 2.5% isoflurane for 6 h (N = 6). * P < 0.05 vs. carrier gas group treated with IgG isotype antibody. # P < 0.05 vs. isoflurane group treated with IgG isotype antibody. Error bars represent 1 SD. TGF-β1 antibody (10 μg/ml) prevents isoflurane-mediated induction of IL-11 mRNA and protein expression in human proximal tubule cells.

Article Snippet: Human and mouse proximal tubule cell culture and exposure to isoflurane Immortalized human renal proximal tubule (HK-2) cells (American Type Culture Collection, Manassas, VA) were grown and passaged with 50:50 mixture of Dulbecco’s Modified Eagle Media/F12 with 10% fetal bovine serum (Invitrogen, Carlsbad, CA) and antibiotics (100 U/ml penicillin G, 100 μg/ml streptomycin, and 0.25 μg/ml amphotericin B (Invitrogen) at 37°C in a 100% humidified atmosphere of 5% carbon dioxide-95% air.

Techniques: Reverse Transcription, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Expressing, Enzyme-linked Immunosorbent Assay

Journal: Anesthesiology

Article Title: Critical Role of Interleukin-11 in Isoflurane-mediated Protection against Ischemic Acute Kidney Injury in Mice

doi: 10.1097/ALN.0b013e3182a950da

Figure Lengend Snippet: IL-11 mRNA (reverse transcription polymerase chain reaction (RTPCR)) expression in primary culture of mouse proximal tubule cells treated with 2.5% isoflurane for 6 h (N = 4). Representative images (top) and band intensity quantifications (bottom) expressed as fold increases in IL-11 expression over carrier gas and immunoglobulin G (IgG) isotype antibody treated controls. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA expression was also quantified to normalize lane loading. * P < 0.05 vs. carrier gas group treated with IgG isotype antibody. # P < 0.05 vs. isoflurane group treated with IgG isotype antibody. Error bars represent 1 SD. TGF-β1 antibody (10 μg/ml) prevents isoflurane-mediated induction of IL-11 mRNA and protein expression in mouse proximal tubule cells.

Article Snippet: Human and mouse proximal tubule cell culture and exposure to isoflurane Immortalized human renal proximal tubule (HK-2) cells (American Type Culture Collection, Manassas, VA) were grown and passaged with 50:50 mixture of Dulbecco’s Modified Eagle Media/F12 with 10% fetal bovine serum (Invitrogen, Carlsbad, CA) and antibiotics (100 U/ml penicillin G, 100 μg/ml streptomycin, and 0.25 μg/ml amphotericin B (Invitrogen) at 37°C in a 100% humidified atmosphere of 5% carbon dioxide-95% air.

Techniques: Reverse Transcription, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Expressing

Journal: Anesthesiology

Article Title: Critical Role of Interleukin-11 in Isoflurane-mediated Protection against Ischemic Acute Kidney Injury in Mice

doi: 10.1097/ALN.0b013e3182a950da

Figure Lengend Snippet: Naïve IL-11 wild-type (WT) mice were exposed to pentobarbital (PB) or 1.2% isoflurane (ISO) for 4 h. A. Representative bands for IL-11 mRNA (reverse transcription polymerase chain reaction (RTPCR)) expression in mouse kidney (N = 4). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as an internal loading control. B. Kidney lysate IL-11 protein (detected by enzyme-linked immunosorbent assay (ELISA)) in IL-11 naïve WT mice exposed to pentobarbital (PB) or 1.2% isoflurane (ISO) for 4 h (N = 4). To neutralize TGF-β1 in vivo, some IL-11 WT mice were injected with 5 mg/kg monoclonal anti-TGF-β1 (MAB240) antibody intravenous injection. TGF-β1 neutralization prevented the induction of IL-11 after isoflurane anesthesia. * P < 0.05 vs. pentobarbital anesthetized mice treated with immunoglobulin G (IgG) isotype antibody. # P < 0.05 vs. isoflurane anesthetized mice treated with IgG isotype antibody. Error bars represent 1 SD. Isoflurane anesthesia significantly increased kidney IL-11 mRNA and protein expression in mice.

Article Snippet: Human and mouse proximal tubule cell culture and exposure to isoflurane Immortalized human renal proximal tubule (HK-2) cells (American Type Culture Collection, Manassas, VA) were grown and passaged with 50:50 mixture of Dulbecco’s Modified Eagle Media/F12 with 10% fetal bovine serum (Invitrogen, Carlsbad, CA) and antibiotics (100 U/ml penicillin G, 100 μg/ml streptomycin, and 0.25 μg/ml amphotericin B (Invitrogen) at 37°C in a 100% humidified atmosphere of 5% carbon dioxide-95% air.

Techniques: Reverse Transcription, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Expressing, Control, Enzyme-linked Immunosorbent Assay, In Vivo, Injection, Neutralization

Journal: Anesthesiology

Article Title: Critical Role of Interleukin-11 in Isoflurane-mediated Protection against Ischemic Acute Kidney Injury in Mice

doi: 10.1097/ALN.0b013e3182a950da

Figure Lengend Snippet: A. IL-11 immunohistochemistry (400× shown, N = 4) in kidneys from IL-11 receptor wild type (IL-11R WT) mice anesthetized with pentobarbital or with 1.2% isoflurane for 4 h. IL-11 staining was darker in kidneys of mice anesthetized with isoflurane. TGF-β1 neutralizing antibody attenuated isoflurane-mediated increase in IL-11 immunoreactivity. Finally, IL-11 staining was not visible in kidneys from mice stained with negative isotype control antibodies (representative of four experiments). B. Quantifications of renal tubular IL-11 staining in mice anesthetized with pentobarbital or with 1.2% isoflurane for 4 h. Kidney IL-11 immunoreactivity significantly increased in mice anesthetized with isoflurane and attenuated with TGF-β1 neutralizing antibody. # P < 0.05 vs. isoflurane anesthetized mice treated with immunoglobulin G (IgG) isotype antibody. * P < 0.05 vs. pentobarbital-anesthetized mice. Error bars represent 1 SD.

Article Snippet: Human and mouse proximal tubule cell culture and exposure to isoflurane Immortalized human renal proximal tubule (HK-2) cells (American Type Culture Collection, Manassas, VA) were grown and passaged with 50:50 mixture of Dulbecco’s Modified Eagle Media/F12 with 10% fetal bovine serum (Invitrogen, Carlsbad, CA) and antibiotics (100 U/ml penicillin G, 100 μg/ml streptomycin, and 0.25 μg/ml amphotericin B (Invitrogen) at 37°C in a 100% humidified atmosphere of 5% carbon dioxide-95% air.

Techniques: Immunohistochemistry, Staining, Control

Journal: Anesthesiology

Article Title: Critical Role of Interleukin-11 in Isoflurane-mediated Protection against Ischemic Acute Kidney Injury in Mice

doi: 10.1097/ALN.0b013e3182a950da

Figure Lengend Snippet: Plasma creatinine levels from IL-11 wild-type (WT) or IL-11 deficient (KO) mice subjected to 30 min renal ischemia and 24 h reperfusion (N = 4-6 per group). After renal ischemia reperfusion (RIR), mice were further anesthetized with 1.2% isoflurane (ISO) or with equi-anesthetic dose of pentobarbital (PB). Some IL-11 receptor (IL-11R) WT mice were pretreated with an IL-11 neutralizing antibody (1 mg/kg, intravenous injection) 20 min before reperfusion or sham-operation. Isoflurane postconditioning significantly reduced plasma creatinine after RIR injury in IL-11R WT mice. However, IL-11R deficiency or IL-11 neutralizing antibody prevented the renal protective effects of isoflurane post-conditioning. * P < 0.05 vs. respective sham-operated mice. # P < 0.05 vs. pentobarbital anesthetized mice subjected to RIR. Data are from 6 mice per group and represented as mean ± SD.

Article Snippet: Human and mouse proximal tubule cell culture and exposure to isoflurane Immortalized human renal proximal tubule (HK-2) cells (American Type Culture Collection, Manassas, VA) were grown and passaged with 50:50 mixture of Dulbecco’s Modified Eagle Media/F12 with 10% fetal bovine serum (Invitrogen, Carlsbad, CA) and antibiotics (100 U/ml penicillin G, 100 μg/ml streptomycin, and 0.25 μg/ml amphotericin B (Invitrogen) at 37°C in a 100% humidified atmosphere of 5% carbon dioxide-95% air.

Techniques: Clinical Proteomics, Injection

Journal: Anesthesiology

Article Title: Critical Role of Interleukin-11 in Isoflurane-mediated Protection against Ischemic Acute Kidney Injury in Mice

doi: 10.1097/ALN.0b013e3182a950da

Figure Lengend Snippet: A. Representative photomicrographs of five to six experiments for hematoxylin and eosin staining (magnification 200×) of kidneys of IL-11 receptor wild-type (IL-11R WT) mice, IL-11 receptor deficient (IL-11R KO) mice and IL-11R WT mice pretreated with IL-11 neutralizing antibody and subjected to 30 min renal ischemia and 24-h reperfusion (I/R). B. Summary of Jablonski scale renal injury scores (N = 4, graded from hematoxylin and eosin staining, scale 0-4) for mice subjected to renal I/R. * P < 0.05 vs. pentobarbital-anesthetized IL-11R WT mice subjected to renal I/R. Error bars represent 1 SD. IL-11R WT mice anesthetized with pentobarbital after renal ischemia showed severe renal tubular necrosis. Isoflurane post-conditioning significantly attenuated renal tubular necrosis and renal injury scores after renal IR. IL-11R deficiency (IL-11R KO) or IL-11 neutralization prevented renal protection with isoflurane postconditioning in mice.

Article Snippet: Human and mouse proximal tubule cell culture and exposure to isoflurane Immortalized human renal proximal tubule (HK-2) cells (American Type Culture Collection, Manassas, VA) were grown and passaged with 50:50 mixture of Dulbecco’s Modified Eagle Media/F12 with 10% fetal bovine serum (Invitrogen, Carlsbad, CA) and antibiotics (100 U/ml penicillin G, 100 μg/ml streptomycin, and 0.25 μg/ml amphotericin B (Invitrogen) at 37°C in a 100% humidified atmosphere of 5% carbon dioxide-95% air.

Techniques: Staining, Neutralization

Journal: Anesthesiology

Article Title: Critical Role of Interleukin-11 in Isoflurane-mediated Protection against Ischemic Acute Kidney Injury in Mice

doi: 10.1097/ALN.0b013e3182a950da

Figure Lengend Snippet: A. Representative photomicrographs of four to six experiments for immunohistochemistry (brown staining) for neutrophil infiltration (200×) from kidneys IL-11 receptor wild-type (IL-11R WT) mice, IL-11 receptor deficient (IL-11R KO) mice and IL-11R WT mice pretreated with IL-11 neutralizing antibody and subjected to 30 min. renal ischemia and 24-h reperfusion. B. Quantifications of infiltrated neutrophils per 200× field in the kidneys of mice after renal ischemia reperfusion (RIR). * P < 0.05 vs. vehicle-treated pentobarbital anesthetized mice subjected to RIR. Error bars represent 1 SD. IL-11R WT mice anesthetized with pentobarbital after renal ischemia showed heavy neutrophil infiltration. Isoflurane postconditioning significantly attenuated renal tubular neutrophil infiltration after RIR. IL-11 deficiency (IL-11R KO) or IL-11 neutralization attenuated these reductions in renal neutrophil infiltration with isoflurane post-conditioning in mice.

Article Snippet: Human and mouse proximal tubule cell culture and exposure to isoflurane Immortalized human renal proximal tubule (HK-2) cells (American Type Culture Collection, Manassas, VA) were grown and passaged with 50:50 mixture of Dulbecco’s Modified Eagle Media/F12 with 10% fetal bovine serum (Invitrogen, Carlsbad, CA) and antibiotics (100 U/ml penicillin G, 100 μg/ml streptomycin, and 0.25 μg/ml amphotericin B (Invitrogen) at 37°C in a 100% humidified atmosphere of 5% carbon dioxide-95% air.

Techniques: Immunohistochemistry, Staining, Neutralization

Journal: Anesthesiology

Article Title: Critical Role of Interleukin-11 in Isoflurane-mediated Protection against Ischemic Acute Kidney Injury in Mice

doi: 10.1097/ALN.0b013e3182a950da

Figure Lengend Snippet: A. Representative photomicrographs of four to six experiments for terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining (representing apoptotic nuclei, magnification 100×) from kidneys IL-11 receptor wild-type (IL-11R WT) mice, IL-11 receptor deficient (IL-11R KO [knockout]) mice and IL-11R WT mice pretreated with IL-11 neutralizing antibody and subjected to 30 min renal ischemia and 24-h reperfusion. B. Quantifications of apoptotic cells per 100× field in the kidneys of mice after renal ischemia reperfusion (RIR). * P < 0.05 vs. vehicle-treated pentobarbital anesthetized mice subjected to RIR. Error bars represent 1 SD. IL-11R WT mice anesthetized with pentobarbital after renal ischemia showed numerous TUNEL positive cells. Isoflurane postconditioning significantly attenuated renal tubular apoptosis after RIR. IL-11 deficiency (IL-11R KO) or IL-11 neutralization attenuated these reductions in renal tubular apoptosis with isoflurane postconditioning in mice.

Article Snippet: Human and mouse proximal tubule cell culture and exposure to isoflurane Immortalized human renal proximal tubule (HK-2) cells (American Type Culture Collection, Manassas, VA) were grown and passaged with 50:50 mixture of Dulbecco’s Modified Eagle Media/F12 with 10% fetal bovine serum (Invitrogen, Carlsbad, CA) and antibiotics (100 U/ml penicillin G, 100 μg/ml streptomycin, and 0.25 μg/ml amphotericin B (Invitrogen) at 37°C in a 100% humidified atmosphere of 5% carbon dioxide-95% air.

Techniques: TUNEL Assay, Staining, Knock-Out, Neutralization

Journal: Anesthesiology

Article Title: Critical Role of Interleukin-11 in Isoflurane-mediated Protection against Ischemic Acute Kidney Injury in Mice

doi: 10.1097/ALN.0b013e3182a950da

Figure Lengend Snippet: Collectively, our data suggest that isoflurane anesthesia increases interleukin (IL)-11 messenger RNA (mRNA) and protein synthesis via TGF-β1 signaling. We propose that IL-11 synthesized then subsequently activates IL-11R in neighboring renal tubules, or endothelial cells to induce cytoprotective signaling. Since previous studies have shown that IL-11 reduces the activity of a well-known proinflammatory transcription factor NF-kB49;50, it is highly possible that IL-11 generated with isoflurane treatment may also attenuate NF-kB activity to protect against renal inflammation and injury after acute kidney injury. SMADs are intracellular proteins that transduce extracellular signals from TGF-β1 to the nucleus to initiate downstream gene transcription. Hypothetical pathways (e.g., NF-kB inhibition) leading to cytoprotection are shown in dashed lines. We previous showed that IL-11 produces renal protection by direct induction of sphingosine kinase-1 via nuclear translocation of HIF-1α.14

Article Snippet: Human and mouse proximal tubule cell culture and exposure to isoflurane Immortalized human renal proximal tubule (HK-2) cells (American Type Culture Collection, Manassas, VA) were grown and passaged with 50:50 mixture of Dulbecco’s Modified Eagle Media/F12 with 10% fetal bovine serum (Invitrogen, Carlsbad, CA) and antibiotics (100 U/ml penicillin G, 100 μg/ml streptomycin, and 0.25 μg/ml amphotericin B (Invitrogen) at 37°C in a 100% humidified atmosphere of 5% carbon dioxide-95% air.

Techniques: Synthesized, Activity Assay, Generated, Inhibition, Translocation Assay

Journal: Frontiers in Immunology

Article Title: Evolutionary and functional characterization of lagomorph guanylate-binding proteins: a story of gain and loss and shedding light on expression, localization and innate immunity-related functions

doi: 10.3389/fimmu.2024.1303089

Figure Lengend Snippet: Differential mRNA expression levels for rabbit GBPs. (A) Heat map of RT-qPCR mRNA expression analysis of ocGBPs and ocFurin in several tissues, primary cells, cell lines and overexpression in RK13 cells: ΔCt values to the reference gene ActinB are displayed (CtGBP – CtActB). Tissues of four female New Zealand white rabbits and three primary cells, cell lines and overexpression were analyzed. Scale: from red (low ΔCt value, i.e., higher expression of target gene) to blue (higher ΔCt, i.e., lower expression of target gene). (B) Rabbit transcriptome was retrieved from and blasted for GBP mRNA expression using the BLAST tool from NCBI. Gray color means present, white means absent.

Article Snippet: SIRC (Cornea, ATCC CCL-60), RAB-9 (Skin, ATCC CRL-1414), RK13 (Kidney, ATCC CCL-37) and RL-33 (Lung, tebu-bio JCRB0131) cell lines were cultured in monolayers in MEM GlutaMAXTM (Gibco) supplemented with 10% heat-inactivated fetal calf serum (FCS, Sigma Aldrich) and 1% Penicillin/Streptomycin (P/S, Sigma Aldrich).

Techniques: Expressing, Quantitative RT-PCR, Over Expression

Journal: Frontiers in Immunology

Article Title: Evolutionary and functional characterization of lagomorph guanylate-binding proteins: a story of gain and loss and shedding light on expression, localization and innate immunity-related functions

doi: 10.3389/fimmu.2024.1303089

Figure Lengend Snippet: Protein expression of overexpressed rabbit GBPs in a rabbit cell line. (A) RK13 cells were transfected with ocGBP expression plasmids. Two days post-transfection, cells were permeabilized and protein expression was determined via flow cytometry. Shown are the mean fluorescence intensities (MFI ± SD) of HA-positive cells stained with PB-coupled antibodies (n = 3). (B) RK13 cells were transfected with rabbit GBP expression plasmids. Two days post-transfection, protein expression was determined using Western blot. Membranes were probed for HA tag (GBP) and Vinculin (housekeeping protein). Shown is a representative Western blot. PB, pacific blue.

Article Snippet: SIRC (Cornea, ATCC CCL-60), RAB-9 (Skin, ATCC CRL-1414), RK13 (Kidney, ATCC CCL-37) and RL-33 (Lung, tebu-bio JCRB0131) cell lines were cultured in monolayers in MEM GlutaMAXTM (Gibco) supplemented with 10% heat-inactivated fetal calf serum (FCS, Sigma Aldrich) and 1% Penicillin/Streptomycin (P/S, Sigma Aldrich).

Techniques: Expressing, Transfection, Flow Cytometry, Fluorescence, Staining, Western Blot

Journal: Frontiers in Immunology

Article Title: Evolutionary and functional characterization of lagomorph guanylate-binding proteins: a story of gain and loss and shedding light on expression, localization and innate immunity-related functions

doi: 10.3389/fimmu.2024.1303089

Figure Lengend Snippet: Intracellular localization of ocGBPs in RK13. RK13 cells were transfected with GBP expression plasmids. Two days post-transfection, localization was determined via immunofluorescence microscopy. The following colors were used: pink (phalloidin, actin filaments), yellow (TGN46, trans-Golgi network), indigo (Hoechst, Nucleus), green (HA-tag, GBPs). Shown are representative images out of 5 -10 imaged positions. 100x magnification, scale bars indicate 10 µm.

Article Snippet: SIRC (Cornea, ATCC CCL-60), RAB-9 (Skin, ATCC CRL-1414), RK13 (Kidney, ATCC CCL-37) and RL-33 (Lung, tebu-bio JCRB0131) cell lines were cultured in monolayers in MEM GlutaMAXTM (Gibco) supplemented with 10% heat-inactivated fetal calf serum (FCS, Sigma Aldrich) and 1% Penicillin/Streptomycin (P/S, Sigma Aldrich).

Techniques: Transfection, Expressing, Immunofluorescence, Microscopy

Journal: The Journal of Biological Chemistry

Article Title: An immunoaffinity-based method for isolating ultrapure adult astrocytes based on ATP1B2 targeting by the ACSA-2 antibody

doi: 10.1074/jbc.M116.765313

Figure Lengend Snippet: Genes cloned for overexpression in a HEK293T system Single cells identified as astrocytes on the basis of established marker genes ( 19 ) were analyzed to identify transcripts fulfilling the following criteria. First, transcripts had to be present in ≥90% of identified astrocytes. Second, transcripts had to encode proteins containing a predicted transmembrane region. Third, only proteins localizing to the plasma membrane were considered. Only proteins fulfilling these criteria could realistically be used in the immunoisolation of live (non-fixed) astrocytes. This table lists identified transcripts fulfilling these criteria and the corresponding PubMed accession numbers of the transcripts used to design subsequent cloning steps. Essentially, PCR primers were designed to flank ORFs of genes of interest (capital letters) as well as to incorporate unique restriction sites on both ends (lowercase letters). Sequences were then amplified by PCR before ligation into the pCAGIG plasmid. Plasmids were verified by sequencing and checked against the deposited amino acid sequence for the respective proteins. Single-nucleotide polymorphisms that did not affect the amino acid sequence were tolerated. In addition to encoding genes of interest, these constructs also encoded cytosolic GFP to act as a marker for cell transfection.

Article Snippet: Overexpression of ORF-encoding plasmids, ACSA-2 staining, and flow cytometry Heterologous expression of identified genes in a HEK293T system HEK293T (human embryonic kidney) cells (sourced from ATCC) were maintained according to standard protocols ( 49 ) and were mycoplasma-free.

Techniques: Clone Assay, Over Expression, Marker, Clinical Proteomics, Membrane, Cloning, Amplification, Ligation, Plasmid Preparation, Sequencing, Construct, Transfection

Journal: The Journal of Biological Chemistry

Article Title: An immunoaffinity-based method for isolating ultrapure adult astrocytes based on ATP1B2 targeting by the ACSA-2 antibody

doi: 10.1074/jbc.M116.765313

Figure Lengend Snippet: Flow cytometry-based experiments identify ATP1B2 as a target for ACSA-2. HEK293T cells (which do not bind ACSA-2 under normal conditions) were transfected with plasmids encoding for proteins identified by our bioinformatic screen (see Table 3). These plasmids also expressed soluble GFP as a marker for successful plasmid transfection. Cells were then stained with an ACSA-2-APC conjugate and analyzed by flow cytometry. From the list of proteins identified in Table 3, only cells expressing ATP1B2 showed strong co-labeling for ACSA-2 and GFP (28.2% of cells). A representative experiment is presented in the figure. This experiment was repeated twice using independent samples (effectively 1 technical replicate per sample) on separate days with the same results. At least 60,000 cells were analyzed per sample. Lines in each plot delineate gates; numbers represent the proportion of cells in each particular gate.

Article Snippet: Overexpression of ORF-encoding plasmids, ACSA-2 staining, and flow cytometry Heterologous expression of identified genes in a HEK293T system HEK293T (human embryonic kidney) cells (sourced from ATCC) were maintained according to standard protocols ( 49 ) and were mycoplasma-free.

Techniques: Flow Cytometry, Transfection, Marker, Plasmid Preparation, Staining, Expressing, Labeling

Journal: Cell Cycle

Article Title: Peroxisomal ACBD4 interacts with VAPB and promotes ER-peroxisome associations

doi: 10.1080/15384101.2017.1314422

Figure Lengend Snippet: . ACBD4iso2 is a peroxisomal C-tail-anchored protein. (A) Schematic overview of ACBD4iso2 domain structure. ACBD = acyl-CoA binding domain, FFAT-like = 2 phenyalanines in an acidic tract, CC = coiled coil, TMD = transmembrane domain. (B-E) Subcellular localization patterns for ACBD4iso2. COS-7 cells transfected with Myc-ACBD4iso2 were immunolabelled using αPEX14 (peroxisomal marker), αTOM20 (mitochondrial marker) and αMyc antibodies. (E) Higher magnifications of boxed regions are shown (F-G) Differential permeabilisation. COS-7 cells expressing FLAG-ACBD4iso2 were fixed, permeabilised with either Triton X-100 (0.2% in PBS) (F) or digitonin (2.5µg/ml in PBS) (G), and stained with αCatalase (PO matrix), αPEX14 (PO membrane) or αFLAG antibodies. Bars, 10 µm (overlay), 2µm (magnified sections).

Article Snippet: COS-7 cells (African green monkey kidney cells; ATCC, https://www.lgcstandards-atcc.org/products/All/CRL-1651) were cultured in DMEM, high glucose (4.5 g/L) supplemented with 10% FBS, 100 U/ml penicillin and 100 µg/ml streptomycin at 37°C with 5% CO 2 and 95% humidity.

Techniques: Binding Assay, Transfection, Marker, Expressing, Staining

Journal: Cell Cycle

Article Title: Peroxisomal ACBD4 interacts with VAPB and promotes ER-peroxisome associations

doi: 10.1080/15384101.2017.1314422

Figure Lengend Snippet: . ACBD4iso2 interacts with VAPB. (A) Identification of VAPB and VAPA by MS after co-immunoprecipitation (IP) with GFP-ACBD4iso2 from COS-7 cells (results from 3 experiments); GFP used as control. Only protein IDs which did not appear in any of the GFP only control experiments were considered. (B) Immunoprecipitation (IP) of GFP-ACBD4iso2 and Myc-VAPB after co-expression in COS-7 cells. GFP used as a negative control and GFP-ACBD5 as a positive control. Samples were immunoprecipitated (GFP-Trap) and immunoblotted (IB) using Myc/GFP antibodies.

Article Snippet: COS-7 cells (African green monkey kidney cells; ATCC, https://www.lgcstandards-atcc.org/products/All/CRL-1651) were cultured in DMEM, high glucose (4.5 g/L) supplemented with 10% FBS, 100 U/ml penicillin and 100 µg/ml streptomycin at 37°C with 5% CO 2 and 95% humidity.

Techniques: Immunoprecipitation, Expressing, Negative Control, Positive Control

Journal: Cell Cycle

Article Title: Peroxisomal ACBD4 interacts with VAPB and promotes ER-peroxisome associations

doi: 10.1080/15384101.2017.1314422

Figure Lengend Snippet: . ACBD4iso2/VAPB co-expression promotes PO-ER association. COS-7 cells were transfected with (A) Myc-VAPB alone (immunolabelled using αPEX14, a peroxisomal marker), (B) Myc-VAPB co-expressed with GFP-ACBD4iso2, (C) Myc-VAPB co-expressed with GFP-ACBD4iso2 showing mitochondrial mistargeting. (D) Co-localization of GFP-ACBD4iso2 with Tom20 (mitochondrial marker). Arrows highlight PO-ER association. Bars, 20 µm (overview), 5 µm (cut outs).

Article Snippet: COS-7 cells (African green monkey kidney cells; ATCC, https://www.lgcstandards-atcc.org/products/All/CRL-1651) were cultured in DMEM, high glucose (4.5 g/L) supplemented with 10% FBS, 100 U/ml penicillin and 100 µg/ml streptomycin at 37°C with 5% CO 2 and 95% humidity.

Techniques: Expressing, Transfection, Marker