Journal: The Journal of cell biology

Article Title: ARFRP1 functions upstream of ARL1 and ARL5 to coordinate recruitment of distinct tethering factors to the trans-Golgi network.

doi: 10.1083/jcb.201905097

Figure Lengend Snippet: Figure 2. Both ARL5 and ARFRP1 are required for localization of GARP to the TGN. (A) KO of TGN-localized small GTPases in HeLa cells confirmed by immunoblot analysis with antibodies to the proteins indicated on the right. In this figure and subsequent figures, ARL5 KO represents KO of both ARL5A and ARL5B, and RAB6 KO represents KO of both RAB6A and RAB6B. α-Tubulin was used as a loading control. The positions of molecular mass markers are indicated on the left. (B) Immunofluorescence microscopy of WT and KO HeLa cells transfected with a plasmid encoding VPS54-13Myc and stained for the Myc epitope (red), giantin (green), and nuclei (DAPI; blue). Scale bars: 10 μm. Insets are magnified views of the boxed areas. Inset scale bars: 5 μm. (C) Quantification of the percentage of cells exhibiting VPS54-13Myc staining at the TGN. Values are the mean ± SEM from three independent experiments. More than 100 cells per sample were counted in each experiment. The statistical significance of the differences relative to WT cells was determined using Dunnett’s test. **, P < 0.01; ***, P < 0.001.

Article Snippet: The following antibodies were used for immunoblotting and/or immunofluorescence microscopy: rabbit anti-VPS51 (HPA039650; Atlas Antibodies), rabbit anti-VPS52 made in our laboratory (Pérez-Victoria et al., 2008), rabbit anti-VPS53 (HPA024446; Atlas Antibodies), mouse anti-VPS50 (FLJ20097, monoclonal antibody M01, 2D11; Abnova), mouse anti-Myc epitope (9E10; Santa Cruz Biotechnology), sheep anti-TGN46 (AHP500G; Bio-Rad), mouse anti–β-actin (G043; Applied Biological Materials), rabbit antigiantin (ab80864; Abcam), mouse HRP-conjugated anti-α-tubulin (DM1A; Santa Cruz Biotechnology), rabbit anti-ARL1 (16012-1-AP; Proteintech), rabbit anti-ARFRP1 (PA5-50606; Thermo Fisher Scientific), mouse anti-ARL5A (sc-514680; Santa Cruz Biotechnology), rabbit anti-RAB6A (GTX110646; GeneTex), rabbit anti-GCC88 (HPA021323; Sigma-Aldrich), rabbit anti-GCC185 (HPA035849; Sigma-Aldrich), mouse anti-Golgin-245 (611281; BD Biosciences), mouse anti-GM130 (610822; BD Biosciences), mouse anti–Golgin-97 (A-21270; Thermo Fisher Scientific), rabbit anti-TMF1 (HPA008729, Sigma-Aldrich), monoclonal HRP-conjugated anti-GFP (Miltenyi Biotec Inc.), rabbit anti-GFP (A-11122; Thermo Fisher Scientific), HRP-conjugated goat anti-rabbit and donkey anti-mouse antibodies (Jackson ImmunoResearch), HRP-conjugated donkey antisheep (R&D Systems), and Alexa Fluor–conjugated secondary antibodies for immunostaining (Thermo Fisher Scientific).

Techniques: Western Blot, Control, Immunofluorescence, Microscopy, Transfection, Plasmid Preparation, Staining

Journal: The Journal of cell biology

Article Title: ARFRP1 functions upstream of ARL1 and ARL5 to coordinate recruitment of distinct tethering factors to the trans-Golgi network.

doi: 10.1083/jcb.201905097

Figure Lengend Snippet: Figure 5. Small GTPases required for localization of golgins to the TGN. (A) Immunofluorescence microscopy of WT, ARL1-KO, ARFRP1-KO, ARL5-KO, and RAB6-KO cells immunostained for endogenous Golgin-245, Golgin-97, GCC88, GCC185, or TMF1 and counterstained with DAPI (blue). Scale bars: 10 μm. Insets are magnified views of the boxed areas. Inset scale bars: 5 μm. Notice that ARL1 KO or ARFRP1 KO caused complete disappearance of Golgin-245 and GCC88, and a partial decrease in the intensity of Golgin-97, at the TGN; quantification in 10 cells per sample in three independent experiment showed that Golgin-97 decrease was 75.6% ± 2.4% in ARL1-KO cells and 55.0% ± 1.9% in ARFRP1-KO cells. (B) SDS-PAGE and immunoblot analysis of endogenous golgins and α-tubulin (loading control) in WT and KO cells. The positions of molecular mass markers are indicated on the left. (C) Immunofluorescence microscopy of RAB6- KO cells transfected with a plasmid encoding GFP-tagged mouse Rab6A (green), immunostained for endogenous GCC185 and TMF1 (red), and counterstained with DAPI (blue). Cells were examined for GFP fluorescence by confocal microscopy. Scale bars: 10 μm.

Article Snippet: The following antibodies were used for immunoblotting and/or immunofluorescence microscopy: rabbit anti-VPS51 (HPA039650; Atlas Antibodies), rabbit anti-VPS52 made in our laboratory (Pérez-Victoria et al., 2008), rabbit anti-VPS53 (HPA024446; Atlas Antibodies), mouse anti-VPS50 (FLJ20097, monoclonal antibody M01, 2D11; Abnova), mouse anti-Myc epitope (9E10; Santa Cruz Biotechnology), sheep anti-TGN46 (AHP500G; Bio-Rad), mouse anti–β-actin (G043; Applied Biological Materials), rabbit antigiantin (ab80864; Abcam), mouse HRP-conjugated anti-α-tubulin (DM1A; Santa Cruz Biotechnology), rabbit anti-ARL1 (16012-1-AP; Proteintech), rabbit anti-ARFRP1 (PA5-50606; Thermo Fisher Scientific), mouse anti-ARL5A (sc-514680; Santa Cruz Biotechnology), rabbit anti-RAB6A (GTX110646; GeneTex), rabbit anti-GCC88 (HPA021323; Sigma-Aldrich), rabbit anti-GCC185 (HPA035849; Sigma-Aldrich), mouse anti-Golgin-245 (611281; BD Biosciences), mouse anti-GM130 (610822; BD Biosciences), mouse anti–Golgin-97 (A-21270; Thermo Fisher Scientific), rabbit anti-TMF1 (HPA008729, Sigma-Aldrich), monoclonal HRP-conjugated anti-GFP (Miltenyi Biotec Inc.), rabbit anti-GFP (A-11122; Thermo Fisher Scientific), HRP-conjugated goat anti-rabbit and donkey anti-mouse antibodies (Jackson ImmunoResearch), HRP-conjugated donkey antisheep (R&D Systems), and Alexa Fluor–conjugated secondary antibodies for immunostaining (Thermo Fisher Scientific).

Techniques: Immunofluorescence, Microscopy, SDS Page, Western Blot, Control, Transfection, Plasmid Preparation, Fluorescence, Confocal Microscopy

Journal: Journal of Cell Science

Article Title: Ezrin directly interacts with AQP2 and promotes its endocytosis

doi: 10.1242/jcs.204842

Figure Lengend Snippet: VP treatment enhances apical AQP2 expression and its colocalization with ezrin. (A) VP treatment enhances apical AQP2 expression and its colocalization with ezrin in cultured renal epithelial cells. AQP2-MDCK cells were stained with antibodies against ezrin (green) and AQP2 (red) in the presence (VP) and absence (Control) of VP treatment (AVP 20 nM for 20 min). The larger panels represent confocal sections through the subapical regions of the cells above the nucleus. The smaller horizontal strips at the bottom of each panel are z-sections through the entire cell for direct comparison of the respective staining intensities of the apical and basolateral membranes, and the cytosol. Upper panels show that in the absence of VP stimulation, ezrin staining localized to the cytosol and basolateral region, while AQP2 staining was mainly detected in the subapical region. Lower panels show that after VP treatment, the ezrin signal was redistributed toward the apical and sub-apical regions and partially colocalized with the similarly apically redistributed AQP2. Scale bar: 10 μm. (B) Super-resolution Airyscan confocal microscopy imaging revealed that AQP2 and ezrin partially colocalize on the apical membrane in VP-treated MDCK cells. Left panels show no apparent colocalization of ezrin and AQP2, in the absence of VP stimulation. Right panels are cells treated with VP. Scale bar: 5 μm. (C) AQP2 and ezrin are co-expressed in principal cells of the Brattleboro rat collecting duct, and co-accumulate on the plasma membrane after vasopressin treatment. Without VP treatment (Control), ezrin was located in the cytosol and basal region, while AQP2 was detected mainly in the sub-apical region of the principal cells of the collecting ducts. After 7 days of VP treatment (VP), ezrin (red in the merge panel) colocalized with AQP2 (green in the merge panel) on the plasma membrane of the principal cells. Scale bar: 20 μm.

Article Snippet: The commercial primary antibodies are from the following vendors: rabbit polyclonal anti-AQP2 antibody (#AQP2-002, Alomone Labs, Jerusalem, Israel), goat polyclonal anti-AQP2 antibody (sc9882, Santa Cruz Biotechnology, Dallas, TX), rabbit polyclonal anti-ezrin (3145s, Cell Signaling), rabbit anti-AQP2 p256 (PhosphoSolutions, Aurora, CO, USA), rabbit anti-AQP2 p261 (PhosphoSolutions), mouse monoclonal anti-GST antibody (sc138, Santa Cruz Biotechnology), mouse monoclonal anti-His antibody (70796, EMD Millipore, Billerica, MA), mouse anti-GAPDH antibody (AM4300, Ambion/Thermo Fisher Scientific), mouse anti β-actin antibody (A5441, Sigma-Aldrich), and rabbit anti-transferrin receptor 1 antibody (ab84036, Abcam, Cambridge, MA).

Techniques: Expressing, Cell Culture, Staining, Confocal Microscopy, Imaging

Journal: Journal of Cell Science

Article Title: Ezrin directly interacts with AQP2 and promotes its endocytosis

doi: 10.1242/jcs.204842

Figure Lengend Snippet: Interaction of AQP2 with ezrin is detected in co-IP experiments. (A) List of ezrin peptides detected by mass spectrometry from the AQP2 co-IP complex. (B,C) By using an anti-ezrin antibody for co-IP, we were able to detect AQP2 in the co-IP complex from stable AQP2-expressing LLC-PK1 cell lysates and mouse kidney (B). Similarly, ezrin signal was detected in the co-IP complex using anti-AQP2 antibody (C). WB, western blotting.

Article Snippet: The commercial primary antibodies are from the following vendors: rabbit polyclonal anti-AQP2 antibody (#AQP2-002, Alomone Labs, Jerusalem, Israel), goat polyclonal anti-AQP2 antibody (sc9882, Santa Cruz Biotechnology, Dallas, TX), rabbit polyclonal anti-ezrin (3145s, Cell Signaling), rabbit anti-AQP2 p256 (PhosphoSolutions, Aurora, CO, USA), rabbit anti-AQP2 p261 (PhosphoSolutions), mouse monoclonal anti-GST antibody (sc138, Santa Cruz Biotechnology), mouse monoclonal anti-His antibody (70796, EMD Millipore, Billerica, MA), mouse anti-GAPDH antibody (AM4300, Ambion/Thermo Fisher Scientific), mouse anti β-actin antibody (A5441, Sigma-Aldrich), and rabbit anti-transferrin receptor 1 antibody (ab84036, Abcam, Cambridge, MA).

Techniques: Co-Immunoprecipitation Assay, Mass Spectrometry, Expressing, Western Blot

Journal: Journal of Cell Science

Article Title: Ezrin directly interacts with AQP2 and promotes its endocytosis

doi: 10.1242/jcs.204842

Figure Lengend Snippet: AQP2 interacts with ezrin via the ezrin N-terminal FERM-containing domain. (A) Recombinant His-tagged ezrin full-length (FL, amino acids 1–586), N-terminus (NT, amino acids 1–308), and C-terminus (CT, amino acids 285–586) were expressed in E. coli and purified to homogeneity as revealed by SDS-PAGE together with purified recombinant AQP2 C-terminus (AQP2CT). Schematic representation of each recombinant protein is shown in B. (C) The purified His-tagged ezrin full-length protein (FL) and the N-terminal FERM-containing recombinant protein (but not the C-terminal protein) were able to pull down AQP2 from both LLC-AQP2 cell lysates and mouse kidney lysates. Lanes 1–3, beads alone pulled down with PBS (lane 1), LLC-AQP2 cell lysate (lane 2) and kidney lysate (lane 3); lanes 4–6, ezrin FL pulled down with PBS (lane 4), LLC-AQP2 cell lysate (lane 5) and kidney lysate (lane 6); lanes 7–9, ezrin NT pulled down with PBS (lane 7), LLC-AQP2 cell lysate (lane 8) and kidney lysate (lane 9); lanes 10–12, ezrin CT pulled down with PBS (lane 10), LLC-AQP2 cell lysate (lane 11) and kidney lysate (lane 12). WB, western blotting.

Article Snippet: The commercial primary antibodies are from the following vendors: rabbit polyclonal anti-AQP2 antibody (#AQP2-002, Alomone Labs, Jerusalem, Israel), goat polyclonal anti-AQP2 antibody (sc9882, Santa Cruz Biotechnology, Dallas, TX), rabbit polyclonal anti-ezrin (3145s, Cell Signaling), rabbit anti-AQP2 p256 (PhosphoSolutions, Aurora, CO, USA), rabbit anti-AQP2 p261 (PhosphoSolutions), mouse monoclonal anti-GST antibody (sc138, Santa Cruz Biotechnology), mouse monoclonal anti-His antibody (70796, EMD Millipore, Billerica, MA), mouse anti-GAPDH antibody (AM4300, Ambion/Thermo Fisher Scientific), mouse anti β-actin antibody (A5441, Sigma-Aldrich), and rabbit anti-transferrin receptor 1 antibody (ab84036, Abcam, Cambridge, MA).

Techniques: Recombinant, Purification, SDS Page, Western Blot

Journal: Journal of Cell Science

Article Title: Ezrin directly interacts with AQP2 and promotes its endocytosis

doi: 10.1242/jcs.204842

Figure Lengend Snippet: AQP2 C-terminus directly interacts with ezrin N-terminal FERM-containing domain. The direct interaction of AQP2 and ezrin is revealed by pulldown experiments using purified recombinant ezrin and AQP2 proteins. Only the purified ezrin full-length (FL) and the N-terminus FERM domain-containing (NT) recombinant protein were able to pull down the purified AQP2 C-terminus. The ezrin C-terminal domain (CT) did not pull down the AQP2 C-terminal domain. WB, western blotting.

Article Snippet: The commercial primary antibodies are from the following vendors: rabbit polyclonal anti-AQP2 antibody (#AQP2-002, Alomone Labs, Jerusalem, Israel), goat polyclonal anti-AQP2 antibody (sc9882, Santa Cruz Biotechnology, Dallas, TX), rabbit polyclonal anti-ezrin (3145s, Cell Signaling), rabbit anti-AQP2 p256 (PhosphoSolutions, Aurora, CO, USA), rabbit anti-AQP2 p261 (PhosphoSolutions), mouse monoclonal anti-GST antibody (sc138, Santa Cruz Biotechnology), mouse monoclonal anti-His antibody (70796, EMD Millipore, Billerica, MA), mouse anti-GAPDH antibody (AM4300, Ambion/Thermo Fisher Scientific), mouse anti β-actin antibody (A5441, Sigma-Aldrich), and rabbit anti-transferrin receptor 1 antibody (ab84036, Abcam, Cambridge, MA).

Techniques: Purification, Recombinant, Western Blot

Journal: Journal of Cell Science

Article Title: Ezrin directly interacts with AQP2 and promotes its endocytosis

doi: 10.1242/jcs.204842

Figure Lengend Snippet: Downregulating ezrin causes membrane accumulation of AQP2. (A) Immunoblotting revealed a strong knockdown of endogenous ezrin in LLC-AQP2 cells by ezrin shRNA lentivirus. There was an ∼80% reduction of endogenous ezrin in ezrin shRNA knockdown LLC-AQP2 cells (graph shows the quantification of ezrin band intensity relative to actin). Results are mean±s.e.m. (n≥3). ***P<0.001 (one-way ANOVA). (B) Immunofluorescence staining of AQP2 in control LLC-AQP2 cells and cells infected with ezrin shRNA lentivirus. After knocking down ezrin in LLC-AQP2 cells, AQP2 was found to increasingly accumulate on the cell surface under baseline conditions (without any stimulation). VP-treated LLC-AQP2 cells were used for comparison. Scale bar: 10 μm. (C) Surface biotinylation experiment revealed a significantly increased accumulation of AQP2 signal on cell surface after knocking down ezrin expression in cells. Transferrin receptor 1 (TFR-1) was used as an internal control for stable membrane proteins. WB, western blotting.

Article Snippet: The commercial primary antibodies are from the following vendors: rabbit polyclonal anti-AQP2 antibody (#AQP2-002, Alomone Labs, Jerusalem, Israel), goat polyclonal anti-AQP2 antibody (sc9882, Santa Cruz Biotechnology, Dallas, TX), rabbit polyclonal anti-ezrin (3145s, Cell Signaling), rabbit anti-AQP2 p256 (PhosphoSolutions, Aurora, CO, USA), rabbit anti-AQP2 p261 (PhosphoSolutions), mouse monoclonal anti-GST antibody (sc138, Santa Cruz Biotechnology), mouse monoclonal anti-His antibody (70796, EMD Millipore, Billerica, MA), mouse anti-GAPDH antibody (AM4300, Ambion/Thermo Fisher Scientific), mouse anti β-actin antibody (A5441, Sigma-Aldrich), and rabbit anti-transferrin receptor 1 antibody (ab84036, Abcam, Cambridge, MA).

Techniques: Western Blot, shRNA, Immunofluorescence, Staining, Infection, Expressing

Journal: Journal of Cell Science

Article Title: Ezrin directly interacts with AQP2 and promotes its endocytosis

doi: 10.1242/jcs.204842

Figure Lengend Snippet: Phosphorylation of key residues in AQP2 was not altered in ezrin knockdown cells. (A) Representative western blot showing that there is no alteration of the levels of total AQP2 and AQP2 phosphorylated at residues 256 or 261 in ezrin shRNA lentivirus-infected LLC-AQP2 cells unlike in cells treated with VP. Immunoblot using anti-β-actin antibody was used as control. Quantification of western blotting results for AQP2 phosphorylated on S256 (p256), S261 (p261) and total AQP2. (B) Intracellular cAMP measurement in LLC-PK1 cells. The intracellular cAMP concentration was significantly increased by 30 min VP treatment (20 nM LVP) without clonal variation. After ezrin knockdown no significant difference in cAMP concentration was observed compared to control. Results in A and B are mean±s.e.m. (n≥3). **P<0.01, ***P<0.001 (one-way ANOVA).

Article Snippet: The commercial primary antibodies are from the following vendors: rabbit polyclonal anti-AQP2 antibody (#AQP2-002, Alomone Labs, Jerusalem, Israel), goat polyclonal anti-AQP2 antibody (sc9882, Santa Cruz Biotechnology, Dallas, TX), rabbit polyclonal anti-ezrin (3145s, Cell Signaling), rabbit anti-AQP2 p256 (PhosphoSolutions, Aurora, CO, USA), rabbit anti-AQP2 p261 (PhosphoSolutions), mouse monoclonal anti-GST antibody (sc138, Santa Cruz Biotechnology), mouse monoclonal anti-His antibody (70796, EMD Millipore, Billerica, MA), mouse anti-GAPDH antibody (AM4300, Ambion/Thermo Fisher Scientific), mouse anti β-actin antibody (A5441, Sigma-Aldrich), and rabbit anti-transferrin receptor 1 antibody (ab84036, Abcam, Cambridge, MA).

Techniques: Western Blot, shRNA, Infection, Concentration Assay

Journal: Journal of Cell Science

Article Title: Ezrin directly interacts with AQP2 and promotes its endocytosis

doi: 10.1242/jcs.204842

Figure Lengend Snippet: Downregulating ezrin reduces clathrin-mediated endocytosis and causes concomitant membrane accumulation of AQP2 without affecting the overall exocytosis. (A) Ezrin knockdown does not affect the overall exocytosis in LLC-AQP2-ssYFP cells. LLC-AQP2 cells were stably transfected with ssYFP. The fluorescence signal in the extracellular medium was measured in LLC-AQP2-ssYFP cells with and without treatment with VP or ezrin shRNA lentivirus, respectively. The fluorescence intensity in the medium reflected the exocytotic activity of LLC-AQP2-ssYFP cells, and therefore the rate of exocytosis. No significant increase was seen in the overall exocytosis in LLC-AQP2-ssYFP cells treated with ezrin shRNA lentivirus compared to control. In contrast, a significant increase in exocytosis was observed in VP-treated LLC-AQP2-ssYFP cells, which is consistent with our previous reports (Nunes et al., 2008). (B) Endocytosis assay using Rhodamine-conjugated transferrin showed that Alexa Fluor 568-labeled transferrin accumulated on the apical membrane following ezrin knockdown (upper panel), and a simultaneous acute membrane accumulation of AQP2 (lower panel). Scale bar: 10 μm. (C) A bar graph showing that ezrin knockdown affects clathrin-mediated endocytosis. A block of endocytosis with MβCD was used as a positive control. Results in A and C are mean±s.e.m. (n≥3). **P<0.01, ***P<0.001 (one-way ANOVA).

Article Snippet: The commercial primary antibodies are from the following vendors: rabbit polyclonal anti-AQP2 antibody (#AQP2-002, Alomone Labs, Jerusalem, Israel), goat polyclonal anti-AQP2 antibody (sc9882, Santa Cruz Biotechnology, Dallas, TX), rabbit polyclonal anti-ezrin (3145s, Cell Signaling), rabbit anti-AQP2 p256 (PhosphoSolutions, Aurora, CO, USA), rabbit anti-AQP2 p261 (PhosphoSolutions), mouse monoclonal anti-GST antibody (sc138, Santa Cruz Biotechnology), mouse monoclonal anti-His antibody (70796, EMD Millipore, Billerica, MA), mouse anti-GAPDH antibody (AM4300, Ambion/Thermo Fisher Scientific), mouse anti β-actin antibody (A5441, Sigma-Aldrich), and rabbit anti-transferrin receptor 1 antibody (ab84036, Abcam, Cambridge, MA).

Techniques: Stable Transfection, Transfection, Fluorescence, shRNA, Activity Assay, Endocytosis Assay, Labeling, Blocking Assay, Positive Control

Journal: Journal of Cell Science

Article Title: Ezrin directly interacts with AQP2 and promotes its endocytosis

doi: 10.1242/jcs.204842

Figure Lengend Snippet: AQP2 internalization was reduced after ezrin knockdown in a cold block experiment. (A) Dynamic distribution of AQP2 after a 20°C cold block was revealed by immunofluorescence staining of LLC-AQP2 cells. Cells were incubated at 20°C for 30, 60 and 120 min to block AQP2 release from the trans-Golgi network. AQP2 formed a ‘perinuclear patch’ in cells at 20°C. In the absence of protein synthesis (blocked by cycloheximide), the speed of formation and immunostaining intensity of the perinuclear patch reflect the speed and extent of AQP2 internalization from the cell surface over time. Scale bar: 10 μm. (B) Quantification of the growth of the AQP2-positive perinuclear patch after 20°C cold block over time. The fluorescence intensity of AQP2 staining in the perinuclear patch was measured using Volocity software as described previously (Arthur et al., 2015). Results are mean±s.e.m. (n≥13 for each data point). Experiments were repeated at least three times.

Article Snippet: The commercial primary antibodies are from the following vendors: rabbit polyclonal anti-AQP2 antibody (#AQP2-002, Alomone Labs, Jerusalem, Israel), goat polyclonal anti-AQP2 antibody (sc9882, Santa Cruz Biotechnology, Dallas, TX), rabbit polyclonal anti-ezrin (3145s, Cell Signaling), rabbit anti-AQP2 p256 (PhosphoSolutions, Aurora, CO, USA), rabbit anti-AQP2 p261 (PhosphoSolutions), mouse monoclonal anti-GST antibody (sc138, Santa Cruz Biotechnology), mouse monoclonal anti-His antibody (70796, EMD Millipore, Billerica, MA), mouse anti-GAPDH antibody (AM4300, Ambion/Thermo Fisher Scientific), mouse anti β-actin antibody (A5441, Sigma-Aldrich), and rabbit anti-transferrin receptor 1 antibody (ab84036, Abcam, Cambridge, MA).

Techniques: Blocking Assay, Immunofluorescence, Staining, Incubation, Immunostaining, Fluorescence, Software

Journal: Journal of Virology

Article Title: PCV2 Induces Reactive Oxygen Species To Promote Nucleocytoplasmic Translocation of the Viral DNA Binding Protein HMGB1 To Enhance Its Replication

doi: 10.1128/JVI.00238-20

Figure Lengend Snippet: PCV2 infection led to translocation of HMGB1 from nuclei to cytoplasmic compartments. PK-15 cells and porcine monocytic cells (3D4/31) were infected for 36 h with PCV2 (MOI = 1) or mock infected as a control. (A) Confocal imaging of HMGB1 distribution in PCV2-infected cells immunostained with anti-HMGB1 (green) and anti-Cap (red) antibodies. Nuclei were labeled with DAPI (blue). Representative micrographic images are shown. (B) Immunoblotting of PCV2 Cap and HMGB1 in nuclear and cytoplasmic extracts from PCV2- or mock-infected PK-15 cells. Histone H3 and GAPDH were used as internal controls for nuclear and cytoplasmic fractions, respectively. (C) The intensity of protein bands was quantified densitometrically using Gel-Pro Analyzer. Ratios of nuclear or cytoplasmic HMGB1 to Histone H3 or GAPDH were quantified, respectively. (D and E) Quantification of hmgb1 mRNA by qPCR in PK-15 and 3D4/31 cells infected with PCV2 for different times using total RNA extracts from the cells. (F and G) Immunoblotting of HMGB1 and PCV2 Cap in the lysates of PK-15 and 3D4/31 cells infected with PCV2 for different times. β-Actin was used as a loading control. The data in panels A, B, F, and G are representative of three independent experiments. Bar charts in panels C, D, and E show means ± SDs from three independent experiments. ns, not significant; *, P < 0.05; **, P < 0.01.

Article Snippet: Approximately 1 mg of total cellular proteins or nuclear proteins was transferred to a 1.5-ml microcentrifuge tube and incubated with 4 μg of rabbit anti-HMGB1 polyclonal antibody (ChIP grade, ab18256) (Abcam) or normal rabbit IgG (Beyotime) for 2 h at 4°C.

Techniques: Infection, Translocation Assay, Imaging, Labeling, Western Blot

Journal: Journal of Virology

Article Title: PCV2 Induces Reactive Oxygen Species To Promote Nucleocytoplasmic Translocation of the Viral DNA Binding Protein HMGB1 To Enhance Its Replication

doi: 10.1128/JVI.00238-20

Figure Lengend Snippet: Overexpression of HMGB1 inhibited PCV2 replication. PK-15 cells were transfected with recombinant plasmid expressing HMGB1 (pHMGB1) or control plasmid (pFlag) for 24 h and then infected with PCV2 (MOI = 1) for 36 h. (A) Effect of HMGB1 overexpression on PCV2 Cap expression as shown by immunoblotting using protein samples from the whole-cell lysates. β-Actin was used as a loading control. The gel shown is representative of three independent experiments. (B) The ratios of band intensity of HMGB1 or PCV2 Cap to β-actin (as shown in panel A). (C) Effect of HMGB1 overexpression on PCV2 orf2 (encoding Cap) transcription measured by qPCR using total RNA extracted from the whole-cell lysates. (D) PCV2 replication in cells overexpressing HMGB1 as assessed by indirect immunofluorescence. Percentages of PCV2-infected cells were calculated as described in the legend for Fig. 2. Relative percentages of PCV2-infected cells in the HMGB1 overexpressing cells are shown with nontransfected but PCV2-infected cells set at 100%. (E) PCV2 genomic DNA copies in cells overexpressing HMGB1 quantified by qPCR using total DNA extracts from whole-cell lysates. Bar charts in panels B, C, D, and E show means ± SDs from three independent experiments. ns, not significant; **, P < 0.01; ***, P < 0.001.

Article Snippet: Approximately 1 mg of total cellular proteins or nuclear proteins was transferred to a 1.5-ml microcentrifuge tube and incubated with 4 μg of rabbit anti-HMGB1 polyclonal antibody (ChIP grade, ab18256) (Abcam) or normal rabbit IgG (Beyotime) for 2 h at 4°C.

Techniques: Over Expression, Transfection, Recombinant, Plasmid Preparation, Expressing, Infection, Western Blot, Immunofluorescence

Journal: Journal of Virology

Article Title: PCV2 Induces Reactive Oxygen Species To Promote Nucleocytoplasmic Translocation of the Viral DNA Binding Protein HMGB1 To Enhance Its Replication

doi: 10.1128/JVI.00238-20

Figure Lengend Snippet: Downregulation of HMGB1 promoted PCV2 replication. PK-15 cells were transfected with hmgb1-specific RNA interference (RNAi) plasmid (sh-HMGB1) or control RNAi plasmid (sh-NC) for 24 h and then infected with PCV2 (MOI= 1) for 36 h. (A) Effect of hmgb1 knockdown on PCV2 Cap expression (β-actin used as a loading control) as shown by immunoblotting using protein samples from the whole-cell lysates. The gel shown is representative of three independent experiments. (B) The ratios of band intensity of HMGB1 or PCV2 Cap to β-actin (as shown in panel A). (C) Effect of hmgb1 knockdown on PCV2 orf2 (encoding Cap) transcription examined by qPCR using total RNA extracted from the whole-cell lysates. (D) PCV2 replication in hmgb1-silenced cells as assessed by indirect immunofluorescence. Representative fluorescence images are shown (top). Percentage of PCV2-infected cells was calculated by dividing the number of PCV2-infected cells by the total cell number in each group (n = 2 images for each experiment per group) that were counted using ImageJ software. Relative percentages of PCV2-infected cells in the hmgb1-silenced cells are shown with nontransfected but PCV2-infected cells set at 100% (bottom). (E) Effect of hmgb1 silencing on PCV2 genomic DNA copies measured by qPCR using total DNA extracts from whole-cell lysates. Bar charts in panels B, C, D, and E show means ± SDs from three independent experiments. ns, not significant; **, P < 0.01.

Article Snippet: Approximately 1 mg of total cellular proteins or nuclear proteins was transferred to a 1.5-ml microcentrifuge tube and incubated with 4 μg of rabbit anti-HMGB1 polyclonal antibody (ChIP grade, ab18256) (Abcam) or normal rabbit IgG (Beyotime) for 2 h at 4°C.

Techniques: Transfection, Plasmid Preparation, Infection, Expressing, Western Blot, Immunofluorescence, Fluorescence, Software

Journal: Journal of Virology

Article Title: PCV2 Induces Reactive Oxygen Species To Promote Nucleocytoplasmic Translocation of the Viral DNA Binding Protein HMGB1 To Enhance Its Replication

doi: 10.1128/JVI.00238-20

Figure Lengend Snippet: Nuclear HMGB1 repressed PCV2 replication. PK-15 cells were transfected with recombinant plasmid expressing HMGB1 (pHMGB1) or control plasmid (pFlag) for 24 h and then infected with PCV2 (MOI = 1) for 36 h. Nuclear and cytoplasmic extracts were prepared for immunoblotting as described in the legend for Fig. 1 Immunoblotting of HMGB1 and PCV2 Cap in the nuclear (A) and cytoplasmic (B) fractions. Histone H3 and GAPDH were used as internal controls for nuclear and cytoplasmic extracts, respectively. Representative images from three independent experiments are shown. The ratios of band intensities of HMGB1 or PCV2 Cap to those of histone H3 (as shown in panel A) in the nuclear fraction (C) or to GAPDH (as shown in panel B) in the cytoplasmic fraction (D). (E) Effect of HMGB1 overexpression on PCV2 orf2 transcription in the nuclei examined by qPCR using total RNA extracted from the nuclear fractions. Results were normalized to histone H3 mRNA in the same samples. (F) PCV2 genomic DNA replication in the nuclei of HMGB1-overexpressing cells quantified by qPCR using total DNA extracted from nuclear fractions. Bar charts in panels C to F show means ± SDs from three independent experiments. ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Article Snippet: Approximately 1 mg of total cellular proteins or nuclear proteins was transferred to a 1.5-ml microcentrifuge tube and incubated with 4 μg of rabbit anti-HMGB1 polyclonal antibody (ChIP grade, ab18256) (Abcam) or normal rabbit IgG (Beyotime) for 2 h at 4°C.

Techniques: Transfection, Recombinant, Plasmid Preparation, Expressing, Infection, Western Blot, Over Expression

Journal: Journal of Virology

Article Title: PCV2 Induces Reactive Oxygen Species To Promote Nucleocytoplasmic Translocation of the Viral DNA Binding Protein HMGB1 To Enhance Its Replication

doi: 10.1128/JVI.00238-20

Figure Lengend Snippet: Ethyl pyruvate inhibited nucleocytoplasmic translocation of HMGB1 in PCV2-infected cells. PK-15 cells were mock infected or infected with PCV2 (MOI = 1) with or without ethyl pyruvate (EP; 7.5 mM) treatment. The cell samples were harvested at 36 hpi. (A) Confocal microscopic images show inhibition of nuclear HMGB1 migration into the cytosol by EP. Cells were immunostained for HMGB1 (green) and PCV2 Cap (red), with nuclei stained with DAPI (blue). Bars, 10 μm. (B) Immunoblotting of HMGB1 and PCV2 Cap in the nuclear and cytoplasmic fractions of PCV2-infected and EP-treated cells. Histone H3 and GAPDH were used as internal controls for nuclear and cytoplasmic extracts, respectively. Representative images from three independent experiments are shown. The ratios of band intensities of HMGB1 or PCV2 Cap to those of histone H3 (as shown in panel B, left) in the nuclear fraction (C) or to GAPDH (as shown in panel B, right) in the cytoplasmic fraction (D). (E) Effect of EP on PCV2 genomic DNA replication by qPCR using DNA extracted from nuclei of PCV2-infected cells treated with 7.5 mM EP. Bar charts in panels C, D, and E show means ± SDs from three independent experiments. ns, not significant; **, P < 0.01.

Article Snippet: Approximately 1 mg of total cellular proteins or nuclear proteins was transferred to a 1.5-ml microcentrifuge tube and incubated with 4 μg of rabbit anti-HMGB1 polyclonal antibody (ChIP grade, ab18256) (Abcam) or normal rabbit IgG (Beyotime) for 2 h at 4°C.

Techniques: Translocation Assay, Infection, Inhibition, Migration, Staining, Western Blot

Journal: Journal of Virology

Article Title: PCV2 Induces Reactive Oxygen Species To Promote Nucleocytoplasmic Translocation of the Viral DNA Binding Protein HMGB1 To Enhance Its Replication

doi: 10.1128/JVI.00238-20

Figure Lengend Snippet: N-Acetylcysteine inhibited PCV2-induced HMGB1 translocation from nuclei to cytosol and repressed PCV2 replication. PK-15 cells were mock infected or infected with PCV2 (MOI = 1) for 12 h and then treated with 10 mM N-acetylcysteine (NAC). The cell samples were harvested at 36 hpi. (A) Confocal imaging of HMGB1 distribution in PCV2-infected and NAC-treated cells after the cells were fixed and immunostained for HMGB1 (green) and Cap (red). Nuclei were stained with DAPI (blue). Bars, 10 μm. (B) Blotting of HMGB1 and PCV2 Cap in the nuclear and cytoplasmic extracts of PCV2-infected cells with or without NAC treatment. Histone H3 and GAPDH were used as internal controls for the nuclear and cytoplasmic fractions, respectively. The figure is representative of three independent experiments. The ratios of band intensities of HMGB1 or PCV2 Cap to histone H3 (as shown in panel B, left) in the nuclear fraction (C) or to GAPDH (as shown in panel B, right) in the cytoplasmic fraction (D). (E) Effect of NAC on PCV2 genomic DNA replication by qPCR using DNA extracted from lysates of PCV2-infected cells treated with NAC. (F) Cytosolic ROS levels in PCV2-infected cells with or without treatment by NAC or ethyl pyruvate (EP) as measured by flow cytometry after probing with DCFH-DA. Bar charts in panels C, D, E, and F show means ± SDs from three independent experiments. *, P < 0.05; **, P < 0.01.

Article Snippet: Approximately 1 mg of total cellular proteins or nuclear proteins was transferred to a 1.5-ml microcentrifuge tube and incubated with 4 μg of rabbit anti-HMGB1 polyclonal antibody (ChIP grade, ab18256) (Abcam) or normal rabbit IgG (Beyotime) for 2 h at 4°C.

Techniques: Translocation Assay, Infection, Imaging, Staining, Flow Cytometry

Journal: Journal of Virology

Article Title: PCV2 Induces Reactive Oxygen Species To Promote Nucleocytoplasmic Translocation of the Viral DNA Binding Protein HMGB1 To Enhance Its Replication

doi: 10.1128/JVI.00238-20

Figure Lengend Snippet: Ethyl pyruvate was inhibitory to PCV2 infection. PK-15 cells were mock infected or infected with PCV2 (MOI = 1) with or without ethyl pyruvate (EP; 7.5 mM) treatment. The cell samples were harvested at 36 hpi. (A) Effect of EP on PCV2 replication in PK-15 cells by immunofluorescence. Percentages of PCV2-infected cells were calculated as described in the legend for Fig. 2. Relative percentages of PCV2-infected cells in the EP-treated cells are shown with untreated but PCV2-infected cells set at 100%. (B) Immunoblotting of HMGB1 and PCV2 Cap in whole-cell lysates with β-actin used as a loading control. (C) The ratios of band intensities of HMGB1 or PCV2 Cap to β-actin (as shown in panel B). Bar charts in panels A and C show means ± SDs from three independent experiments. ns, not significant; **, P < 0.01.

Article Snippet: Approximately 1 mg of total cellular proteins or nuclear proteins was transferred to a 1.5-ml microcentrifuge tube and incubated with 4 μg of rabbit anti-HMGB1 polyclonal antibody (ChIP grade, ab18256) (Abcam) or normal rabbit IgG (Beyotime) for 2 h at 4°C.

Techniques: Infection, Immunofluorescence, Western Blot

Journal: Journal of Virology

Article Title: PCV2 Induces Reactive Oxygen Species To Promote Nucleocytoplasmic Translocation of the Viral DNA Binding Protein HMGB1 To Enhance Its Replication

doi: 10.1128/JVI.00238-20

Figure Lengend Snippet: The B box domain of HMGB1 was involved in inhibition of PCV2 replication. (A) Schematic illustration of the full-length and truncated forms of porcine HMGB1 according to its human homolog. All truncated versions, A box, AB box, and B box plus C terminus (B boxCT), were flag tagged. The numbers indicate positions of amino acids. Arrows with C followed by numbers represent key cysteine residues. NLS, nuclear localization signal. PK-15 cells were transfected with recombinant plasmids expressing flag-tagged or full-length HMGB1 for 24 h and then infected with PCV2 (MOI = 1) for 36 h. (B) Numbers of PCV2-infected cells examined by immunofluorescence using anti-Cap monoclonal antibody as the probe (top). Expression of PCV2 Cap and different forms of HMGB1 as assessed by immunoblotting using the whole-cell lysates harvested at 36 hpi and antibodies against Flag, Cap, and HMGB1 (bottom). β-Actin was used as a loading control. The panel B images are representative of three individual experiments. (C) The ratios of band intensities of PCV2 Cap to those of β-actin (as shown at the bottom panel of B). (D) Effect of different HMGB1 truncations on PCV2 DNA replication estimated by qPCR using total DNA extracted from the whole-cell lysate. Bar charts in panels C and D show means ± SDs from three independent experiments. ns, not significant; **, P < 0.01.

Article Snippet: Approximately 1 mg of total cellular proteins or nuclear proteins was transferred to a 1.5-ml microcentrifuge tube and incubated with 4 μg of rabbit anti-HMGB1 polyclonal antibody (ChIP grade, ab18256) (Abcam) or normal rabbit IgG (Beyotime) for 2 h at 4°C.

Techniques: Inhibition, Transfection, Recombinant, Expressing, Infection, Immunofluorescence, Western Blot

Journal: Journal of Virology

Article Title: PCV2 Induces Reactive Oxygen Species To Promote Nucleocytoplasmic Translocation of the Viral DNA Binding Protein HMGB1 To Enhance Its Replication

doi: 10.1128/JVI.00238-20

Figure Lengend Snippet: HMGB1 bound to the Ori region of the PCV2 genome. (A) Binding of porcine HMGB1 to PCV2 DNA using the gel shift assay. PCV2 DNA (500 ng) and various concentrations (0 to 5 μg) of purified His-tagged recombinant HMGB1 were mixed in binding buffer. The DNA-protein mixtures were subjected to 0.8% agarose gel electrophoresis to visualize changes of the DNA motility. (B) Binding of HMGB1 to a specific region of PCV2 DNA: full-length and different fragments of PCV2 genome (orf1, orf2, and Ori) were incubated with recombinant HMGB1 protein to identify the region of PCV2 genome involved in HMGB1 binding. (C) To confirm the Ori region is required for HMGB1 binding, the Ori fragment was combined with orf1 or orf2 (Ori-orf1 or Ori-orf2) that were then compared with orf1 or orf2 alone by the gel shift assay. (D) Immunoprecipitation of purified HMGB1 protein (500 μg) and PCV2 DNA (500 ng) mixture by anti-HMGB1 antibody (rabbit IgG as control) and protein A/G agarose. The precipitates were probed with anti-His and anti-HMGB1 antibodies by immunoblotting. (E) Quantification of PCV2 genomic Ori copies by qPCR in DNA extracts from PCV2 DNA-HMGB1 precipitates (shown in panel D) after DNase pretreatment. (F) Blotting of HMGB1 in immunoprecipitates of whole-cell lysates (WCL) of the PK-15 cells infected with PCV2 (36 h) by anti-HMGB1 (rabbit IgG as control) and protein A/G agarose. (G) Quantification of PCV2 genomic Ori copies by qPCR in DNA extracts from immunoprecipitates of whole-cell lysates (shown in panel F) after DNase pretreatment. (H) Blotting of HMGB1 in immunoprecipitates of nuclear extracts of the PK-15 cells infected with PCV2. (I) Quantification of PCV2 genomic Ori copies in the precipitates shown in panel H. Bar charts in panels E, G, and I show means ± SDs from three independent experiments.

Article Snippet: Approximately 1 mg of total cellular proteins or nuclear proteins was transferred to a 1.5-ml microcentrifuge tube and incubated with 4 μg of rabbit anti-HMGB1 polyclonal antibody (ChIP grade, ab18256) (Abcam) or normal rabbit IgG (Beyotime) for 2 h at 4°C.

Techniques: Binding Assay, Electrophoretic Mobility Shift Assay, Purification, Recombinant, Agarose Gel Electrophoresis, Incubation, Immunoprecipitation, Western Blot, Infection

Journal: Journal of Virology

Article Title: PCV2 Induces Reactive Oxygen Species To Promote Nucleocytoplasmic Translocation of the Viral DNA Binding Protein HMGB1 To Enhance Its Replication

doi: 10.1128/JVI.00238-20

Figure Lengend Snippet: Effect of hydrogen peroxide treatment on subcellular localization of HMGB1 and PCV2 replication. (A) H2O2 treatment promoted nucleocytoplasmic translocation of HMGB1. PK-15 cells were treated with or without N-acetylcysteine (NAC; 10 mM) before adding 50 μM H2O2. Cells were fixed and immunostained with anti-HMGB1 (green) for confocal microscopy. Nuclei were labeled with DAPI (blue). (B) Immunoblotting of HMGB1 in the nuclear and cytoplasmic fractions of PK-15 cells treated with H2O2 and NAC. (C) Confocal imaging of PK-15 cells infected by PCV2 with or without 50 μM H2O2 treatment after immunostaining with anti-HMGB1 (green) and anti-Cap (red) antibodies. (D) Blotting of HMGB1 and PCV2 Cap in the nuclear and cytoplasmic extracts of PCV2-infected cells with or without H2O2 treatment. Histone H3 and GAPDH were used as internal controls for the nuclear and cytoplasmic fractions, respectively. (E) Percentages of PCV2-infected cells were calculated from immunofluorescence images as described in the legend for Fig. 2. Relative percentages of PCV2-infected cells in the H2O2-treated cells are shown with nontreated but PCV2-infected cells set at 100%. Bar chart in panel E shows means ± SDs from three independent experiments. **, P < 0.01.

Article Snippet: Approximately 1 mg of total cellular proteins or nuclear proteins was transferred to a 1.5-ml microcentrifuge tube and incubated with 4 μg of rabbit anti-HMGB1 polyclonal antibody (ChIP grade, ab18256) (Abcam) or normal rabbit IgG (Beyotime) for 2 h at 4°C.

Techniques: Translocation Assay, Confocal Microscopy, Labeling, Western Blot, Imaging, Infection, Immunostaining, Immunofluorescence

Journal: Journal of Virology

Article Title: PCV2 Induces Reactive Oxygen Species To Promote Nucleocytoplasmic Translocation of the Viral DNA Binding Protein HMGB1 To Enhance Its Replication

doi: 10.1128/JVI.00238-20

Figure Lengend Snippet: Schematic illustration of the interaction between PCV2 and HMGB1 in infected cells. HMGB1 in the nucleus restricts PCV2 replication by binding to the Ori region of the PCV2 genome. PCV2 infection causes increased generation of cellular ROS. Increased ROS promotes nucleocytoplasmic translocation of HMGB1 and lessens sequestration of the viral DNA by HMGB1 in the nucleus, thus enhancing PCV2 replication. N-Acetylcysteine (and probably ethyl pyruvate as well) scavenges PCV2-induced ROS and thus increases retention of HMGB1 in the nucleus, leading to sequestration of viral DNA and reduced PCV2 replication.

Article Snippet: Approximately 1 mg of total cellular proteins or nuclear proteins was transferred to a 1.5-ml microcentrifuge tube and incubated with 4 μg of rabbit anti-HMGB1 polyclonal antibody (ChIP grade, ab18256) (Abcam) or normal rabbit IgG (Beyotime) for 2 h at 4°C.

Techniques: Infection, Binding Assay, Translocation Assay

Journal: Molecules

Article Title: Quantification of Trastuzumab–HER2 Engagement In Vitro and In Vivo

doi: 10.3390/molecules25245976

Figure Lengend Snippet: Figure 1. Confocal microscopy of Human EGF Receptor 2 (HER2) and trastuzumab (TZM)–AF700 distribution in AU565 cells. (A) Immunofluorescence analysis of HER2 (green; arrows), early endosomal antigen 1 (EEA1), or transferrin receptor (TfR) (red; arrows). Middle slices of z-stacks consisting of 6–8 optical slices are shown. (B) AU565 cells were loaded with 5 µg/mL TZM–AF700 (red) and 5 µg/mL Tf–AF568 (magenta) for 24 h and then processed for immunostaining of HER2 (green) and endosomal markers EEA1 or TfR (magenta). Maximum intensity projections of z-stacks consisting of 6–8 optical slices are shown. Nuclei are visualized with DAPI. Scale bar = 20 µm.

Article Snippet: Primary antibodies were as followed: rabbit monoclonal HER2 1:800 (Cell Signaling, #2165), rabbit monoclonal HER3 1:250 (Cell Signaling, #12708), rabbit monoclonal TZM 1:100 (R&D Systems, MAB95471-100).

Techniques: Confocal Microscopy, Immunostaining

Journal: Molecules

Article Title: Quantification of Trastuzumab–HER2 Engagement In Vitro and In Vivo

doi: 10.3390/molecules25245976

Figure Lengend Snippet: Figure 2. Comparison of intracellular uptake of TZM–AF700 into HER2-positive and HER2-negative cell lines. (A) To test the specificity of HER2-mediated TZM–AF700 binding and internalization, competition experiments were performed. AU565 cells were loaded with 5 µg/mL TZM–AF700 (red) and 5 µg/mL Tf-AF568 (green) in the presence of increasing amounts of unlabeled TZM. Single slice confocal microscopy displays TZM–AF700 (red) and Tf–AF568 (green) binding and internalization into AU565 cells in the presence of increasing 0×, 10×, 50×, and 100× unlabeled TZM. (B) HER2-positive AU565 and HER2-negative MDA-MB-231 and MCF10A cells were subjected to 24 h uptake of 20 µg/mL TZM–AF700 (red) and then processed for anti-HER2 immunostaining (green). Nuclei were visualized with DAPI (blue). Maximum intensity projections of z-stacks consisting of 6–8 optical slices are shown. Confocal images were collected using identical settings on an LSM880 confocal microscope. Scale bar = 20 µm.

Article Snippet: Primary antibodies were as followed: rabbit monoclonal HER2 1:800 (Cell Signaling, #2165), rabbit monoclonal HER3 1:250 (Cell Signaling, #12708), rabbit monoclonal TZM 1:100 (R&D Systems, MAB95471-100).

Techniques: Comparison, Binding Assay, Confocal Microscopy, Immunostaining, Microscopy

Journal: Molecules

Article Title: Quantification of Trastuzumab–HER2 Engagement In Vitro and In Vivo

doi: 10.3390/molecules25245976

Figure Lengend Snippet: Figure 3. TZM Forster Resonance Energy Transfer (FLI-FRET) microscopy (FLIM-FRET) analysis in AU565 cancer cells. (A) The representative time-correlated single photon counting (TCSPC) images of fluorescence intensity and mean lifetime map (τm) in cells treated with TZM-AF700 only [Acceptor/Donor (A:D) = 0:1; donor single-labeled] or with TZM–AF700 plus TZM–AF750 (A:D = 2:1; double-labeled); pseudo-color range = 500–1500 ps. Zoomed regions of interest (ROIs) of both single and double-labeled cells show heterogeneity of fluorescence lifetime of TZM–AF700 within the cells. White arrows indicate x, y coordinates used for the curve fitting using SPCImage. Scale bar = 50 µm. (B) Representative fitting curves and IRF, the fluorescent lifetime decay in the single and double-labeled cells was determined by comparing the fitting of the decay data using both single- and double-exponential decay models, respectively. (C) Fluorescent lifetime distribution in AU565 cells treated with TZM–AF700 (A:D = 0:1) or TZM–AF700 and TZM–AF750 (A:D = 2:1). (D) Quantification of FRET donor (FD%) levels in AU565 cells treated with a near-infrared (NIR) TZM–FRET pair at various A:D ratios. Analysis was performed using 10 distinct pixel coordinates (n = 10) from five independent ROIs; error bars represent confidence interval at 95%. (E) Quantification of TZM–FRET efficiency (E) in relation to A:D ratios. Data presented as mean ± confidence interval at 95%, n = 10.

Article Snippet: Primary antibodies were as followed: rabbit monoclonal HER2 1:800 (Cell Signaling, #2165), rabbit monoclonal HER3 1:250 (Cell Signaling, #12708), rabbit monoclonal TZM 1:100 (R&D Systems, MAB95471-100).

Techniques: Förster Resonance Energy Transfer, Microscopy, Labeling

Journal: Molecules

Article Title: Quantification of Trastuzumab–HER2 Engagement In Vitro and In Vivo

doi: 10.3390/molecules25245976

Figure Lengend Snippet: Figure 4. TZM FLIM-FRET analysis in MDA-MB-231 cancer cells. (A) The representative TCSPC images of fluorescence intensity and mean lifetime map (τm) in cells treated with TZM–AF700 (A:D = 0:1) or with TZM–AF700 plus TZM–AF750 (A:D = 2:1) pseudo-color range = 300–1500 ps. Scale bar = 50 µm. (B) Comparison of fluorescent lifetime distribution in MDA-MB-231 (solid lines) and AU565 cells (dashed lines) treated with TZM–AF700 (A:D = 0:1 red), TZM–AF700 plus TZM–AF750 (A:D = 2:1 black).

Article Snippet: Primary antibodies were as followed: rabbit monoclonal HER2 1:800 (Cell Signaling, #2165), rabbit monoclonal HER3 1:250 (Cell Signaling, #12708), rabbit monoclonal TZM 1:100 (R&D Systems, MAB95471-100).

Techniques: Comparison

Journal: Molecules

Article Title: Quantification of Trastuzumab–HER2 Engagement In Vitro and In Vivo

doi: 10.3390/molecules25245976

Figure Lengend Snippet: Figure 5. Schematic representation of live small animal NIR wide-field time-resolved macroscopic fluorescence lifetime FRET imaging (MFLI-FRET) for preclinical studies. (A) AU565 tumor xenograft production; (B) Intravenous tail-vein injection of AF700–TZM and/or AF750–TZM; (C) Live small animal imaging using wide-field time-resolved FLI-FRET macroscopy (MFLI-FRET) imager; (D) Ex vivo validation using immunohistochemical (IHC) and H&E staining. Inset top left: illustration of TZM–AF700 and TZM–AF750 ligands. Inset bottom right: illustration of TZM–HER2 FLI-FRET events upon binding of donor- and acceptor-labeled antibodies to HER2 dimer at the surface of cancer cells. DMD: digital micromirror device; CCD: charge-coupled device.

Article Snippet: Primary antibodies were as followed: rabbit monoclonal HER2 1:800 (Cell Signaling, #2165), rabbit monoclonal HER3 1:250 (Cell Signaling, #12708), rabbit monoclonal TZM 1:100 (R&D Systems, MAB95471-100).

Techniques: Imaging, Injection, Ex Vivo, Biomarker Discovery, Immunohistochemical staining, Staining, Binding Assay, Labeling

Journal: Molecules

Article Title: Quantification of Trastuzumab–HER2 Engagement In Vitro and In Vivo

doi: 10.3390/molecules25245976

Figure Lengend Snippet: Figure 6. Whole body quantification of TZM–HER2 engagement via MFLI-FRET in vivo imaging. (A) Mice were injected with 20 µg TZM–AF700 alone (M1) or TZM–AF700 and 40 µg TZM–AF750 and subjected to MFLI-FRET imaging at 48 h p.i. Panels show TZM donor maximum intensity ROIs (both soluble and bound probe) and FRET donor fraction (FD%) map (bound and internalized probe) in the tumors (T), livers (LV), and urinary bladders (UB). (B) Histograms of FD% retrieved for each tumor. Numbers represent mean ±SD. (C) Quantification of FD% in the tumors, bladders and livers. Data presented as box indicating 25–75% pixel values, horizontal and vertical lines indicate mean with ±1.5 SD, respectively.

Article Snippet: Primary antibodies were as followed: rabbit monoclonal HER2 1:800 (Cell Signaling, #2165), rabbit monoclonal HER3 1:250 (Cell Signaling, #12708), rabbit monoclonal TZM 1:100 (R&D Systems, MAB95471-100).

Techniques: In Vivo Imaging, Injection, Imaging

Journal: Molecules

Article Title: Quantification of Trastuzumab–HER2 Engagement In Vitro and In Vivo

doi: 10.3390/molecules25245976

Figure Lengend Snippet: Figure 7. IHC ex vivo validation confirms TZM accumulation in the cancer cells both positive for HER2 and HER3. Consecutive sections of tumors M1–M3 and tumor from untreated mouse were processed for H&E staining, anti-HER2, anti-HER3, and anti-TZM immunohistochemical staining. NovaRED was used as peroxidase substrate (brown stain), tissue was counterstained with methyl green. Scale bar = 100 µm.

Article Snippet: Primary antibodies were as followed: rabbit monoclonal HER2 1:800 (Cell Signaling, #2165), rabbit monoclonal HER3 1:250 (Cell Signaling, #12708), rabbit monoclonal TZM 1:100 (R&D Systems, MAB95471-100).

Techniques: Ex Vivo, Biomarker Discovery, Staining, Immunohistochemical staining

Journal: Biology

Article Title: Transdifferentiation of Human Fibroblasts into Skeletal Muscle Cells: Optimization and Assembly into Engineered Tissue Constructs through Biological Ligands

doi: 10.3390/biology10060539

Figure Lengend Snippet: Determination of efficacy of C2C12 differentiation in conjunction with the exposure to ligand combinations. C2C12s were differentiated for 7 days and treated with combination ligands of GDF11 (G), TMSB4X (T), IL6 (I), and TNF-α (F) at 10 ng/mL for seven additional days. ( A ) Fusion index was calculated from total myotube nuclei vs. total nuclei ( n = 16, mean + SD). ( B ) Multinucleation of C2C12 myotubes were quantified ( n = 11, mean + SD). ( C ) Nuclear density was evaluated from nuclear count per field of 5x microscopy ( n = 4, mean + SD). ( D ) C2C12 exposed to ligand combinations were stained to express nuclear MYOD1 ( n = 6, mean + SD). ( E ) Cells were stained with Ki67, and where similarly quantified based on average total nuclear count ( n = 6, mean + SD). ( F ) ACTN2 and Ki67 immunostaining of control cells. ( G ) Cells exposed to GTF showed decreases in fusion index and myonucleation levels, although no change in nuclear density and Ki67+ expression was detected. ( H ) GTIF supplementation significantly reduced skeletal muscle differentiation parameters fusion index and multinucleation, in addition to decreasing average nuclear density. ( I ) Control C2C12s expressing nuclear MYOD1. ( J ) GTF treatment greatly reduced nuclear fusion and showed limited differentiation capacity while expressing comparable levels of nuclear MYOD1. ( K ) Exposure of C2C12s to GTIF combination significantly inhibited skeletal muscle differentiation. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Proteins utilized included recombinant human Follistatin (Fs, 669-FO-025), recombinant human Myostatin (GDF8, 788-G8-010) or Growth Differentiation factor (GDF8), recombinant human basic Fibroblast Growth Factor 2 (FGF2, 233-FB-025), recombinant human GDF11 (1958-GD-010), recombinant human GDF15 (957-GD-025/CF), recombinant human Bone Morphogenetic Protein 4 (BMP4, 314-BP-010/CF), recombinant human BMP7 (354-BP-010), recombinant human Growth Hormone (hGH, 1067-GH-025), recombinant human Interleukin 6 (IL6, 206-IL-010), recombinant human Tumor Necrosis Factor Alpha (TNF-α, 210-TA-005) (All R&D Systems), and Thymosin β (TOCRIS, 3390).

Techniques: Microscopy, Staining, Immunostaining, Control, Expressing

Journal: Biology

Article Title: Transdifferentiation of Human Fibroblasts into Skeletal Muscle Cells: Optimization and Assembly into Engineered Tissue Constructs through Biological Ligands

doi: 10.3390/biology10060539

Figure Lengend Snippet: Effect of ligand combination exposure on differentiation of skeletal muscle cells derived from tHFs. Cells were transduced with MYOD1 fragments and induced to express the skeletal muscle phenotype via the induction of doxycycline and SB431542 over a 7-day period. Ligand combinations of GDF11 (G), TMSB4X (T), IL6 (I), and TNF-α (F) at 10 ng/mL were introduced for an additional week, and SB and Dox administration was discontinued. Skeletal muscle cells were fixed and stained on day 14 and characterized by various differentiation and proliferation parameters from 5× microscopy. ( A ) Fusion index of tHFs was evaluated by determining the ratio of myotube nuclei vs total nuclear count ( n = 16, mean + SD). ( B ) Cellular multinucleation was quantified to assess tHF development of differentiation ( n = 22, mean + SD). ( C ) Nuclear density was similarly assessed by quantifying nuclear count per field ( n = 4, mean + SD). ( D ) Nuclear MYOD1 was quantified ( n = 6, mean + SD). ( E ) Ki67 nuclei were also assessed with a nuclear count ( n = 6, mean + SD). ( F ) Control tHF myotubes were immunostained with ACTN2 and Ki67. ( G ) IL6 and TNF-α combination demonstrated significant decrease in differentiation parameters fusion index, multinucleation, myotube length, and diameter , although Ki67+ expression had increased. ( H ) Exposure of tHFs to combined GDF11, TMSB4X, IL6, and TNF-α showed similar results, however nuclear Ki67 expression was unchanged. ( I ) Untreated tHFs with ACTN2 and MYOD1 nuclear stains. ( J ) Cells treated with IF showed a decrease in MYOD1 nuclear expression. ( K ) Additionally, GDF11, TMSB4X, and IL6 exposure yielded similar results with respect to MYOD1+. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Proteins utilized included recombinant human Follistatin (Fs, 669-FO-025), recombinant human Myostatin (GDF8, 788-G8-010) or Growth Differentiation factor (GDF8), recombinant human basic Fibroblast Growth Factor 2 (FGF2, 233-FB-025), recombinant human GDF11 (1958-GD-010), recombinant human GDF15 (957-GD-025/CF), recombinant human Bone Morphogenetic Protein 4 (BMP4, 314-BP-010/CF), recombinant human BMP7 (354-BP-010), recombinant human Growth Hormone (hGH, 1067-GH-025), recombinant human Interleukin 6 (IL6, 206-IL-010), recombinant human Tumor Necrosis Factor Alpha (TNF-α, 210-TA-005) (All R&D Systems), and Thymosin β (TOCRIS, 3390).

Techniques: Derivative Assay, Transduction, Staining, Microscopy, Control, Expressing

Journal: Biology

Article Title: Transdifferentiation of Human Fibroblasts into Skeletal Muscle Cells: Optimization and Assembly into Engineered Tissue Constructs through Biological Ligands

doi: 10.3390/biology10060539

Figure Lengend Snippet: Skeletal muscle tissues were engineered from a composite fibrin/Matrigel hydrogel mixture with mouse skeletal myoblasts C2C12s, and subject to 10 ng/mL biological ligands. C2C12s were encapsulated and differentiated in a fibrin-based hybrid hydrogel over a 7-day period, 10 ng/mL biological ligands GDF11, TMSB4X, IL6 or TNF-α were administered after a week of tissue plating. ( A ) Immunohistochemical staining of C2C12 skeletal muscle constructs with ACTN2 and DAPI, demonstrated high cellular density. ( B ) Skeletal myotubes increased compactness and alignment towards central pillar regions where tensile force is maximal ( C ) Structural organization of C2C12s at pillar regions appeared disrupted due to gel contraction. ( D ) Cross-striated, multinucleated skeletal muscle form condensed tissues as demonstrated with high magnification 60× confocal microscopy. ( E ) Myotube diameter (µm) was not affected by one-week exposure to 10 ng/mL ligands. ( n > 32, mean + SD). ( F ) Nuclear density of skeletal muscle C2C12s within tissue were not impacted with ligand administration. ( n = 6, mean + SD).

Article Snippet: Proteins utilized included recombinant human Follistatin (Fs, 669-FO-025), recombinant human Myostatin (GDF8, 788-G8-010) or Growth Differentiation factor (GDF8), recombinant human basic Fibroblast Growth Factor 2 (FGF2, 233-FB-025), recombinant human GDF11 (1958-GD-010), recombinant human GDF15 (957-GD-025/CF), recombinant human Bone Morphogenetic Protein 4 (BMP4, 314-BP-010/CF), recombinant human BMP7 (354-BP-010), recombinant human Growth Hormone (hGH, 1067-GH-025), recombinant human Interleukin 6 (IL6, 206-IL-010), recombinant human Tumor Necrosis Factor Alpha (TNF-α, 210-TA-005) (All R&D Systems), and Thymosin β (TOCRIS, 3390).

Techniques: Immunohistochemical staining, Staining, Construct, Confocal Microscopy

Journal: Medical research archives

Article Title: Identification of amacrine neurons with a glycinergic and GABAergic phenotype in the mouse retina

doi: 10.18103/mra.v10i1.2624

Figure Lengend Snippet: List of antibodies used in this study

Article Snippet: Anti-Mouse (CY3) , Donkey , Jackson Immunoresearch Laboratories, 715–165-150 , .

Techniques:

Journal: Medical research archives

Article Title: Identification of amacrine neurons with a glycinergic and GABAergic phenotype in the mouse retina

doi: 10.18103/mra.v10i1.2624

Figure Lengend Snippet: A) Vertical sections from the central retina of the pGlyT2-eGFP mouse were subjected to immunostaining and analyzed by confocal microscopy. Z-stack images were acquired and an optical section from the middle of the stack selected for presentation. Left panel: bright field showing the retinal layers; middle panel, eGFP expression; right panel, merge image. Scale bar, 20 μm. B) Vertical section stained with anti-Syntaxin 1 (HPC-1) antibodies and DAPI followed by CY-3 labeled secondary antibodies. C) Same as B but sections labeled with rabbit antibodies against GlyT1 or D) anti-glycine. Scale bar, 10 μm. Abbreviations: INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer. E) Quantitation of EGFP-positive or EGFP/GlyT1 neurons. Cell counts were obtained from a minimum of 100 HPC-1 positive cells per retina, for a total of three retinas from three different mice. The plots represent the percent of HPC-1 positive cells +/− SE. Open arrowhead in C and D indicate eGFP labeled cells devoid of GlyT1or Glycine immunoreactivity, respectively.

Article Snippet: Anti-Mouse (CY3) , Donkey , Jackson Immunoresearch Laboratories, 715–165-150 , .

Techniques: Immunostaining, Confocal Microscopy, Expressing, Staining, Labeling, Quantitation Assay

Journal: Medical research archives

Article Title: Identification of amacrine neurons with a glycinergic and GABAergic phenotype in the mouse retina

doi: 10.18103/mra.v10i1.2624

Figure Lengend Snippet: A) Vertical sections from the central retina of the pGlyT2-eGFP mouse were immunolabeled with antibodies recognizing the GABA synthetizing enzyme GAD67 and rabbit GlyT1 antibodies or synaptic systems or followed by the corresponding secondary CY-3 and CY-5 labeled antibodies. Z-stack images were acquired through the corresponding channels and an optical section from the middle of the stack selected and used for this figure panels. B) Sections were stained as in A but using the rabbit GABA transporter anti-GAT1 and rat anti-GlyT1 antibodies or C) GAT3 and rat GlyT1. Scale bar 10 μm. Abbreviations: INL, inner nuclear layer; IPL, inner plexiform layer. Open arrowhead in A-C indicate eGFP labeled cells containing GlyT1 and GAD67, or GAT1 or GAT3. D) A minimum of 100 HPC-1 positive cells per retina from three different eyes were used to quantify the number of GAD67 or GlyT1 positive cells and those containing both markers. The plots represent the percent of GAD67 or/and GlyT1 positive cells +/− SE. E) Same as D but normalized by 100 EGFP positive ACs.

Article Snippet: Anti-Mouse (CY3) , Donkey , Jackson Immunoresearch Laboratories, 715–165-150 , .

Techniques: Immunolabeling, Labeling, Staining

Journal: Medical research archives

Article Title: Identification of amacrine neurons with a glycinergic and GABAergic phenotype in the mouse retina

doi: 10.18103/mra.v10i1.2624

Figure Lengend Snippet: Vertical section of the central mouse retina were labeled with antibodies against the protein gephyrin (upper panel) or the GlyR subunits using the mAb4a antibody that recognizes all the receptor subunits, followed by staining with CY-3 labeled secondary antibodies. Confocal Z-stack images were acquired and an optical section from the middle of the stack selected and used for presentation. Scale bar, 10 μm.

Article Snippet: Anti-Mouse (CY3) , Donkey , Jackson Immunoresearch Laboratories, 715–165-150 , .

Techniques: Labeling, Staining

Journal: Medical research archives

Article Title: Identification of amacrine neurons with a glycinergic and GABAergic phenotype in the mouse retina

doi: 10.18103/mra.v10i1.2624

Figure Lengend Snippet: A) Vertical sections from the central retina of C57BL/6J mice were subjected to immunostaining with GlyT1 and GAD67 antibodies followed by incubation with CY-3 and Alexa-488 labeled secondary antibodies, respectively. Scale bar, 50 μm. A1 and A2) Magnification of the areas enclosed in panel A, cells labeled for GAD67 and GlyT1 are marked by an arrow. B) Vertical sections were stained with HPC-1, GlyT1 and GAD67 and the resulting images used for quantitation as described in ​in4D.4D. C) Vertical sections from the central retina of the transgenic line pGAD67-EGFP were labeled with GlyT1 antibody from Synaptic Systems followed by incubation with CY-3 labeled secondary antibodies. Nuclei were stained with DAPI. Arrows indicate co-expression of GlyT1 and eGFP in amacrine neurons. Scale bar, 10 μm for B and C. D) Sections from the pGAD67-EGFP were subjected to quantitation analysis, as described in ​in4D.4D. Scale bar, 10 μm.

Article Snippet: Anti-Mouse (CY3) , Donkey , Jackson Immunoresearch Laboratories, 715–165-150 , .

Techniques: Immunostaining, Incubation, Labeling, Staining, Quantitation Assay, Transgenic Assay, Expressing

Journal: Medical research archives

Article Title: Identification of amacrine neurons with a glycinergic and GABAergic phenotype in the mouse retina

doi: 10.18103/mra.v10i1.2624

Figure Lengend Snippet: A) Primary dissociated retinal cultures were fixed with paraformaldehyde and incubated with antibodies recognizing GAD67 and GlyT1 followed by staining with the corresponding CY-3 and CY-5 labeled antibodies, respectively. The inhibitory neurons were heterogeneous and contained eGFP and GlyT1 (long arrows), or eGFP, GAD67 and GlyT1 (open arrowheads), or GAD67 (thin open arrows). B) Cells were subjected to immunostaining with antibodies against GAT1 and GlyT1 or C) against GAT3 and GlyT1 as described under Experimental Procedures. Scale bars, 10 μm.

Article Snippet: Anti-Mouse (CY3) , Donkey , Jackson Immunoresearch Laboratories, 715–165-150 , .

Techniques: Incubation, Staining, Labeling, Immunostaining

Journal: The American Journal of Pathology

Article Title: Caveolae Participate in Tumor Necrosis Factor Receptor 1 Signaling and Internalization in a Human Endothelial Cell Line

doi: 10.1016/s0002-9440(10)62346-2

Figure Lengend Snippet: Figure 1. Morphological examination of caveolae in resting EA.hy926 cells. A: Transmission electron microscopy reveals caveolae, indicated by arrow- heads, in resting EA.hy926 cells. B: EM analysis of MCD-treated cells showing the disappearance of caveolae network. C: Confocal immunofluo- rescence microscopy with a combination of anti-TNFR1/caveolin antibodies in both control (top) and MCD-treated (bottom) EA.hy926 cells. White boxes indicate high magnification (right). Note co-localization of TNFR1 and caveolin in the periphery of control but not MCD-treated cells. Data are from two different independent experiments.

Article Snippet: Recombinant human TNF- and goat anti-human TNFR1 antibody were purchased from R&D Systems Inc. (Minneapolis, MN).

Techniques: Transmission Assay, Electron Microscopy, Microscopy, Control

Journal: The American Journal of Pathology

Article Title: Caveolae Participate in Tumor Necrosis Factor Receptor 1 Signaling and Internalization in a Human Endothelial Cell Line

doi: 10.1016/s0002-9440(10)62346-2

Figure Lengend Snippet: Figure 2. Fractionation of EA.hy926 cells on sucrose gradient. A: EA.hy926 cells were harvested and fractionated by sucrose density gradient as de- scribed in Materials and Methods and 10 l of each sample was subjected to a dot-blot analysis by staining membrane with HRP-conjugated cholera toxin subunit B (CTxB). B: EA.hy926 cells were incubated for 30 minutes with two different doses of methyl--cyclodextrin (MCD) or not treated, Dounce homogenized, and then fractionated by sucrose density gradient. Fractions, harvested from the top to the bottom of each gradient were analyzed by SDS-PAGE and immunoblotted for TNFR1 and caveolin-1. Ctr, indicates control cells not subjected to MCD treatment. C: Fractions 3 to 5, pooled after fractionation of resting cells, were subjected to immunoelectron micros- copy analysis with both TNFR1 (15 nm, arrowhead) and caveolin (5 nm, arrow) antibodies. Note co-localization of TNFR1 and caveolae in isolated caveolae.

Article Snippet: Recombinant human TNF- and goat anti-human TNFR1 antibody were purchased from R&D Systems Inc. (Minneapolis, MN).

Techniques: Fractionation, Dot Blot, Staining, Membrane, Incubation, SDS Page, Control, Isolation

Journal: The American Journal of Pathology

Article Title: Caveolae Participate in Tumor Necrosis Factor Receptor 1 Signaling and Internalization in a Human Endothelial Cell Line

doi: 10.1016/s0002-9440(10)62346-2

Figure Lengend Snippet: Figure 5. Co-immunoprecipitation of TNFR1 and caveolin-1 from caveolae in EA.hy926 cells. EA.hy926 cells were stimulated with TNF for the indicated time and fractionated by sucrose gradient as described in Materials and Methods. Four hundred l from fractions 3, 4, and 5 (left), and from 9, 10, and 11 were mixed and immunoprecipitated with TNFR1 followed by SDS- PAGE analysis with both TNFR1 and caveolin-1 antibodies. Note that al- though both the proteins are represented in fractions 9 to 11 (see Figure 4A), no interaction between the two proteins was detected. Immunoprecipitation of TNFR1 performed in resting cells not subjected to the fractionation pro- cedure, shows the ability of MCD to disrupt the association between TNFR1 and caveolin-1, as indicated by R at the left. Data are from one of three independent experiments with similar results.

Article Snippet: Recombinant human TNF- and goat anti-human TNFR1 antibody were purchased from R&D Systems Inc. (Minneapolis, MN).

Techniques: Immunoprecipitation, SDS Page, Fractionation

Journal: The American Journal of Pathology

Article Title: Caveolae Participate in Tumor Necrosis Factor Receptor 1 Signaling and Internalization in a Human Endothelial Cell Line

doi: 10.1016/s0002-9440(10)62346-2

Figure Lengend Snippet: Figure 6. TNFR1 does not internalize in clathrin-coated structures in EA.hy926 cells. A: EA.hy926 cells were either left untreated or treated for different periods of time with 10 ng/ml of TNF as indicated and analyzed by laser confocal microscopy after immunostaining with a combination of goat anti-human TNFR1 and mouse anti-clathrin heavy chain antibodies. B: Con- focal analysis of clathrin and transferrin receptor (TfR) in resting EA.hy926 cells. Right: Higher magnification (indicated by box) of the overlay image. Images shown are representative of three independent experiments with similar results. Original magnifications, 63.

Article Snippet: Recombinant human TNF- and goat anti-human TNFR1 antibody were purchased from R&D Systems Inc. (Minneapolis, MN).

Techniques: Confocal Microscopy, Immunostaining

Journal: The American Journal of Pathology

Article Title: Caveolae Participate in Tumor Necrosis Factor Receptor 1 Signaling and Internalization in a Human Endothelial Cell Line

doi: 10.1016/s0002-9440(10)62346-2

Figure Lengend Snippet: Figure 7. TNF-induces trafficking of TNFR1 to endosomes in EA.hy926 cells. EA.hy926 cells were treated with 10 ng/ml of TNF and subjected to confocal fluorescent microscopy by staining with a combination of TNFR1 with either EEA1 (A) or Rab5 (B) antibodies. Images shown are from one of three different experiments with similar results.

Article Snippet: Recombinant human TNF- and goat anti-human TNFR1 antibody were purchased from R&D Systems Inc. (Minneapolis, MN).

Techniques: Microscopy, Staining

Journal: The FASEB Journal

Article Title: Loss of Nfat5 promotes lipid accumulation in vascular smooth muscle cells

doi: 10.1096/fj.202100682r

Figure Lengend Snippet: FIGURE 2 Analysis of the responses of human VSMCs to cholesterol stimulation. Human VSMCs were treated with cholesterol- supplemented medium (Panserin, chol:MbCD 10 µg/mL) for three days. The expression of genes encoding determinants of cholesterol and phospholipid processing was analyzed by semi-quantitative PCR (A, **P < .01, *P < .05, n = 6, the expression of the housekeeping gene RPL32 served as internal standard). NFAT5 was detected by immunostaining. NFAT5-positive nuclei were detected by immunofluorescence-based methods and quantified by automated image analysis (B, ***P < .001, n = 10, scale bar: 50 µm). NFAT5 protein abundance was determined in nuclear and cytosolic fractions of cell lysates by immunoblot techniques (C and D, *P < .05, n = 6, histone H3 and α-tubulin served as loading controls for the nuclear and cytosolic fractions, respectively. Ctr., control; Chol., cholesterol)

Article Snippet: Cells were incubated with a rabbit anti- NFAT5 antibody (NB120- 3446; Novus Biologicals) at 4°C overnight.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Immunostaining, Immunofluorescence, Quantitative Proteomics, Western Blot, Control

Journal: The FASEB Journal

Article Title: Loss of Nfat5 promotes lipid accumulation in vascular smooth muscle cells

doi: 10.1096/fj.202100682r

Figure Lengend Snippet: FIGURE 3 Analysis of the responses of mouse aortic VSMCs to cholesterol stimulation. VSMCs isolated from aortae of Nfat5fl/fl mice were exposed to cholesterol-supplemented medium (Panserin, chol:MbCD 10 µg/mL) or the respective solvent control for three days. Abca1, Acat1, Hmgcr, Lgals3, and Cd68 expression was analyzed by real-time quantitative PCR (A). The expression of the housekeeping gene Rps12 served as internal control (A, *P < .05, ***P < .001 vs control, n = 6-7). NFAT5 protein was detected by capillary electrophoresis in cytosolic and nuclear fractions of cell lysates B and C. HDAC1 and α-tubulin served as loading controls for the nuclear and cytosolic fractions respectively (C, Cytosol: n.s. vs control; Nucleus: *P < .05 vs control, n = 3, Ctr., control; Chol., cholesterol; n.s., not significant)

Article Snippet: Cells were incubated with a rabbit anti- NFAT5 antibody (NB120- 3446; Novus Biologicals) at 4°C overnight.

Techniques: Isolation, Solvent, Control, Expressing, Real-time Polymerase Chain Reaction, Electrophoresis

Journal: The FASEB Journal

Article Title: Loss of Nfat5 promotes lipid accumulation in vascular smooth muscle cells

doi: 10.1096/fj.202100682r

Figure Lengend Snippet: FIGURE 4 Genetic ablation of Nfat5 and its effect on cholesterol-exposed VSMCs. Genetic ablation of Nfat5 was achieved by expressing Cre-recombinase in murine Nfat5fl/fl VSMCs via adenoviral transduction (AdCre, an empty vector adenovirus (AdPl) served as control). The knockout of Nfat5 was evidenced by real-time quantitative PCR analysis (A, ***P < .001 vs control, n = 6, Rps12 expression level served as reference) and confirmed on protein level (B, results from capillary electrophoresis and automated signal detection, Valosin-containing protein (VCP) served as loading reference). VSMCs were exposed to cholesterol- supplemented medium for three days (Panserin, chol:MbCD 10 µg/ mL) 24 h after viral transduction which did not alter the expression of genes associated with a foam-cell like phenotype (C, n.s. vs control, n = 6, Chol., cholesterol; n.s., not significant)

Article Snippet: Cells were incubated with a rabbit anti- NFAT5 antibody (NB120- 3446; Novus Biologicals) at 4°C overnight.

Techniques: Expressing, Transduction, Plasmid Preparation, Control, Knock-Out, Real-time Polymerase Chain Reaction, Electrophoresis

Journal: The FASEB Journal

Article Title: Loss of Nfat5 promotes lipid accumulation in vascular smooth muscle cells

doi: 10.1096/fj.202100682r

Figure Lengend Snippet: FIGURE 5 Identification of Nfat5-controlled arteriosclerosis-associated transcriptional targets. The Nfat5-regulated expression of selected transcripts associated with the development of arteriosclerotic plaques was analyzed by applying a macroarray-based PCR screening protocol comparing cholesterol-exposed murine Nfat5fl/fl and Nfat5−/− VSMCs. Transcripts which were at least four-fold (rounded up) down- regulated in Nfat5-deficient VSMCs were summarized (A, significantly regulated transcripts were marked in yellow and their individual results were shown in B, *P < .05 vs control, n = 3, Ctr., control; Chol., cholesterol; Hsp90ab1 served as reference; comparable results were obtained if Gapdh was chosen as reference). The functional effect of the Nfat5 knockout was analyzed by comparing the cholesterol accumulation in cholesterol-exposed murine Nfat5fl/fl and Nfat5−/− VSMCs (C, Oil Red O (ORO)-stained area, **P < .01 vs. control, one out of two independent experiments with comparable results analyzing 37-62 cells per group is shown, scale bar: 50 µm). Additionally, the cellular cholesterol content was assessed by utilizing a direct colorimetric cholesterol assay (D, *P < .05 vs control, n = 4). Lipids were also visualized in murine aortic VSMCs overexpressing NFAT5 (AdN5; control: AdPl) after exposure to cholesterol (E, ***P < .001 vs. control, one out of three independent experiments with comparable results determining the ORO-positive area of 37-39 cells, scale bars: 50 µm)

Article Snippet: Cells were incubated with a rabbit anti- NFAT5 antibody (NB120- 3446; Novus Biologicals) at 4°C overnight.

Techniques: Expressing, Control, Functional Assay, Knock-Out, Staining, Cholesterol Assay

Journal: The FASEB Journal

Article Title: Loss of Nfat5 promotes lipid accumulation in vascular smooth muscle cells

doi: 10.1096/fj.202100682r

Figure Lengend Snippet: FIGURE 6 Analyses of blood cholesterol levels and lipid accumulation in the aorta of mice fed an atherogenic diet upon SMC-specific genetic ablation of Nfat5. Nfat5fl/fl (N5fl/fl) and Nfat5(SMC)−/− (N5(SMC)−/−) mice were fed an atherogenic diet (AD) for either 14 or 25 weeks or a normal chow diet (ND). Blood cholesterol levels were significantly elevated in mice fed an atherogenic diet (A, *P < .05 vs N5fl/fl ND, n = 4-8; **P < .01 vs N5(SMC)−/− ND, n = 6-9). Aortae were excised and stained with Oil Red O (ORO) to detect lipid-containing areas (B, scale bars: 1 mm). The cumulative ORO-stained vessel area was recorded via light microscopy and quantified (C, ***P < .001, **P < .01, *P < .05 vs control, n = 5-9, representative images are shown in D) and the location of ORO-positive areas in Nfat5(SMC)−/− mice was mapped (E). Confocal microscopy-based analyses of whole mount preparations of these aortae revealed ORO-positive droplets in the subintimal layers (F, scale bars: 100 and 40 µm)

Article Snippet: Cells were incubated with a rabbit anti- NFAT5 antibody (NB120- 3446; Novus Biologicals) at 4°C overnight.

Techniques: Staining, Light Microscopy, Control, Confocal Microscopy