Journal: Arthritis & rheumatology (Hoboken, N.J.)

Article Title: Podocyte Activation of NLRP3 Inflammasomes Contributes to the Development of Proteinuria in Lupus Nephritis

doi: 10.1002/art.40155

Figure Lengend Snippet: A, Representative Western blot bands (left) and relative expression levels in each group (right) showed the expression of NLRP3 and caspase-1 p20, normalized to the values of GAPDH and caspase-1 in the kidneys of 36-week-old NZM2328 mice versus 12-week-old NZM2328 mice. B, Levels of IL-1β in renal extracts of NZM2328 mice. Glomeruli were isolated from NZM2328 mice and single cell suspensions were prepared. Flow cytometry analysis was used to assess the active caspase-1 levels in podocytes (C) and endothelial cells (D). Results are mean ± SD. *p<0.05, **p<0.01, NS = no significance, n=3 per group.

Article Snippet: For the staining of human kidney sections, Alexa Flour A647-conjugated anti-synaptopodin Ab (Santa Cruz), FAM-FLICA Caspase-1 Assay kit and Alexa Flour A555 conjugated mouse mAb against human IL-1β (R&D Systems) were used.

Techniques: Western Blot, Expressing, Isolation, Flow Cytometry

Journal: Arthritis & rheumatology (Hoboken, N.J.)

Article Title: Podocyte Activation of NLRP3 Inflammasomes Contributes to the Development of Proteinuria in Lupus Nephritis

doi: 10.1002/art.40155

Figure Lengend Snippet: Human kidney biopsies were collected and prepared for immunofluorescence staining. Anti- synaptopodin Ab (red), FAM-FLICA Caspase-1 assay kit (green) and mAb anti-human IL-1β (green) were used to detect expression of active caspase-1 and IL-1β in podocytes in the human kidneys. (A) Normal kidney isolated from non-disease area of nephrectomy specimen from a patient with renal cell cancer. (B) Class IV LN biopsy and (C) Class V LN biopsy. Original magnification ×400.

Article Snippet: For the staining of human kidney sections, Alexa Flour A647-conjugated anti-synaptopodin Ab (Santa Cruz), FAM-FLICA Caspase-1 Assay kit and Alexa Flour A555 conjugated mouse mAb against human IL-1β (R&D Systems) were used.

Techniques: Immunofluorescence, Staining, Expressing, Isolation

Journal: Arthritis & rheumatology (Hoboken, N.J.)

Article Title: Podocyte Activation of NLRP3 Inflammasomes Contributes to the Development of Proteinuria in Lupus Nephritis

doi: 10.1002/art.40155

Figure Lengend Snippet: Fresh urine was collected and cells were spun onto a slide by a Cytospin. They were fixed and stained with anti- synaptopodin Ab (red), FAM-FLICA Caspase-1 assay kit (green) and mAb anti-human IL-1β (green) to detect expression of active caspase-1 and IL-1β in urine podocytes. DAPI (blue) was used to stain nuclei. (A) Expression of caspase 1 in the urinary podoycyte in a patient with LN but not in that of a normal individual. (B) Detection of IL-1β in the urine podocytes in a patient with LN but not from a normal control. Original magnification ×630.

Article Snippet: For the staining of human kidney sections, Alexa Flour A647-conjugated anti-synaptopodin Ab (Santa Cruz), FAM-FLICA Caspase-1 Assay kit and Alexa Flour A555 conjugated mouse mAb against human IL-1β (R&D Systems) were used.

Techniques: Staining, Expressing

Journal: Arthritis & rheumatology (Hoboken, N.J.)

Article Title: Podocyte Activation of NLRP3 Inflammasomes Contributes to the Development of Proteinuria in Lupus Nephritis

doi: 10.1002/art.40155

Figure Lengend Snippet: Following 6 weeks’ treatment of MCC950, mouse kidneys were collected for the following analysis. A–C, the kidneys were subjected to Western blot (representative bands shown) for expression of caspase 1 and its active form caspase-1 p20. D, levels of IL-1β in kidney extract determined by ELISA. E, anti-synaptopodin Ab (red) and FAM-FLICA Caspase-1 assay kit were used to detect caspase-1 expression (green) in glomerular podocytes, cell nuclei were marked by DAPI (blue). Original magnification ×400. After 2 weeks’ treatment, flow cytometry analysis was used to assess the active caspase-1 levels in podocytes (F) and endothelial cells (G). Results are mean ± SD. *P<0.05, **P<0.01, n=8 per group.

Article Snippet: For the staining of human kidney sections, Alexa Flour A647-conjugated anti-synaptopodin Ab (Santa Cruz), FAM-FLICA Caspase-1 Assay kit and Alexa Flour A555 conjugated mouse mAb against human IL-1β (R&D Systems) were used.

Techniques: Western Blot, Expressing, Enzyme-linked Immunosorbent Assay, Flow Cytometry

Journal: Arthritis & rheumatology (Hoboken, N.J.)

Article Title: Podocyte Activation of NLRP3 Inflammasomes Contributes to the Development of Proteinuria in Lupus Nephritis

doi: 10.1002/art.40155

Figure Lengend Snippet: The NZM2328 podocyte cell line was stimulated with a control serum or a serum from diseased NZM2328 mice or serum from diseased NZM2328 mice depleted of IgG, with or without MCC950 or Mito TEMPO for 24 hours. A, Representative Western blot (left) and relative expression levels (right) in each group showed the expression of caspase-1 p20, normalized to the values of caspase-1. B, Levels of IL-1β in supernatant following different stimulation. Flow cytometry analysis was used to assess active caspase-1 levels (C), production of ROS (D) and mitochondrial membrane potential (E). F, Expression of P2X7R detected by Western blot. G, Caspase-1 p20 expression analysis followed stimulation with diseased serum or diseased serum depleted of IgG. Results were mean ± SD. *p<0.05, **p<0.01, NS = no significance.

Article Snippet: For the staining of human kidney sections, Alexa Flour A647-conjugated anti-synaptopodin Ab (Santa Cruz), FAM-FLICA Caspase-1 Assay kit and Alexa Flour A555 conjugated mouse mAb against human IL-1β (R&D Systems) were used.

Techniques: Western Blot, Expressing, Flow Cytometry, Membrane

Journal: Scientific Reports

Article Title: Establishment and characterization of an orthotopic patient-derived Group 3 medulloblastoma model for preclinical drug evaluation

doi: 10.1038/srep46366

Figure Lengend Snippet: ( a ) MYC expression is demonstrated in primary tumour and xenografts with in situ hybridization. Scale bar is 20 μm. Western blotting detecting c-Myc ( b ) and cyclin B1 ( c ) in protein extracts isolated from MB-LU-181 cells JQ1 treatment. SK-N-AS neuroblastoma cells and human dermal fibroblasts (HDF) cells were included as positive and negative control for c-Myc expression, respectively. β-actin was used to ensure equal sample loading. ( d ) Cell viability measurement using WST-1 of ( d ) MB-LU-181 and DAOY cells treated with the indicated concentrations of JQ1 for 72-hours. IC 50 values between MB-LU-181 and DAOY cells were significantly different ( p < 0.0001 t-test; 0.27 μM in MB-LU-181 versus 10 μM in DAOY). Mean with SD from three independent experiments are displayed.

Article Snippet: After membrane blocking, the incubation with primary antibody was carried out with c-Myc (D84C12) Rabbit mAb (#5605, Cell Signaling, Danvers, USA) (dilution 1/1000), cyclin B1 Mouse mAb (sc-245, Santa Cruz Biotechnology, Santa Cruz, USA) (dilution 1/500), and β-Actin mouse mAb (#3700, Cell Signaling, Danvers, USA) (dilution 1/10,000), followed by incubation with a peroxidase-conjugated goat anti-rabbit antibody (#7074, Cell Signaling, Danvers, USA) (dilution 1/5,000) or horse anti-mouse (#7076, Cell Signaling, Danvers, USA) (dilution 1/4000 for cyclin B1 and 1/20,000 for β-Actin).

Techniques: Expressing, In Situ Hybridization, Western Blot, Isolation, Negative Control

Journal: Journal of Virology

Article Title: Human Papillomavirus Major Capsid Protein L1 Remains Associated with the Incoming Viral Genome throughout the Entry Process

doi: 10.1128/JVI.00537-17

Figure Lengend Snippet: L1 protein associated with condensed chromosomes retains the reactivity of the H16.V5 epitope. (A and B) At 24 hpi, HaCaT cells infected with EdU-labeled pseudovirus were fixed, permeabilized, and treated with or without Click-iT reaction buffer without dye. Next, the cells were incubated with L1 conformationally dependent mouse MAb H16.V5 antibody (green) and AF488-conjugated anti-α-tubulin (white). Lastly, the cells were treated with Click-iT reaction buffer with AF555 dye (red) to stain the EdU-labeled pseudogenome and mounted in DAPI (blue). Note that colocalization of the EdU puncta and L1 protein is denoted as a yellow color.

Article Snippet: The microtubule network was detected using mouse MAb anti-α-tubulin conjugated to Alexa Fluor 488 (AF488; catalog no. 8058S; Cell Signaling).

Techniques: Infection, Labeling, Incubation, Staining

Journal: Journal of Virology

Article Title: Human Papillomavirus Major Capsid Protein L1 Remains Associated with the Incoming Viral Genome throughout the Entry Process

doi: 10.1128/JVI.00537-17

Figure Lengend Snippet: L1 protein associated with condensed chromosomes retains the reactivity of the H16.56E epitope. (A and B) At 24 hpi, HaCaT cells infected with EdU-labeled pseudovirus were fixed, permeabilized, and treated with or without Click-iT reaction buffer without dye. Next, the cells were incubated with L1 conformationally dependent mouse MAb H16.56E antibody (green) and AF488-conjugated anti-α-tubulin (white). Lastly, the cells were treated with Click-iT reaction buffer with AF555 dye (red) to stain the EdU-labeled pseudogenome and mounted in DAPI (blue). Note that colocalization of the EdU puncta and L1 protein is denoted as a yellow color.

Article Snippet: The microtubule network was detected using mouse MAb anti-α-tubulin conjugated to Alexa Fluor 488 (AF488; catalog no. 8058S; Cell Signaling).

Techniques: Infection, Labeling, Incubation, Staining

Journal: Journal of Virology

Article Title: Human Papillomavirus Major Capsid Protein L1 Remains Associated with the Incoming Viral Genome throughout the Entry Process

doi: 10.1128/JVI.00537-17

Figure Lengend Snippet: The linear epitope of the 33L1-7 antibody is hidden in the L1 protein associated with condensed chromosomes. (A and B) At 24 hpi, HaCaT cells infected with EdU-labeled pseudovirus were fixed, permeabilized, and treated with or without with Click-iT reaction buffer without dye. Next, the cells were incubated with mouse MAb 33L1-7 antibody (green) and AF488-conjugated anti-α-tubulin (white). Lastly, the cells were treated with Click-iT reaction buffer with AF555 dye (red) to stain the EdU-labeled pseudogenome and mounted in DAPI (blue). Note that colocalization of the EdU puncta and L1 protein is denoted as a yellow color.

Article Snippet: The microtubule network was detected using mouse MAb anti-α-tubulin conjugated to Alexa Fluor 488 (AF488; catalog no. 8058S; Cell Signaling).

Techniques: Infection, Labeling, Incubation, Staining

Journal: Journal of Virology

Article Title: Human Papillomavirus Major Capsid Protein L1 Remains Associated with the Incoming Viral Genome throughout the Entry Process

doi: 10.1128/JVI.00537-17

Figure Lengend Snippet: Some full-length L1 protein accompanies the viral genome to the nucleus. (A) HeLa cells were infected with HPV16 pseudovirus with or without 1.5 μM Eg5 inhibitor III (Eg5i) and tracked via live-cell imaging using the IncuCyte Zoom for 48 h. Note that the images are depicted at 18 hpi. (B) HaCaT cells were infected with EdU-labeled HPV16 pseudovirus in the presence of 1.5 μM Eg5i. The cells were fixed at 24 hpi and permeabilized with either digitonin at 0.625 μg/ml or 0.5% TX-100 and then treated with AF555 (green) in Click-iT reaction buffer. The cells were permeabilized again with 0.5% TX-100 and treated with AF647 (red) in Click-iT reaction buffer. Lastly, the cells were mounted in DAPI (blue). (C) HaCaT cells were infected with EdU-labeled pseudovirus and treated with 1.5 μM Eg5i. At 24 hpi, the cells were fixed and permeabilized in 0.5% TX-100. Next, the cells were treated with AF555 (red) in Click-iT reaction buffer, followed by incubation with AF488-conjugated anti-α-tubulin (white) and MAb 33L1-7 (green). Lastly, the cells were mounted in DAPI (blue). Note the colocalization between EdU and L1 signal denoted by white arrows. (D) HeLa cells were infected with HPV16 pseudovirus in the presence of 1.5 μM Eg5i. Cells were trypsinized for 2 min, and monoastral cells were collected. Next, the cells were treated with 15 μl of 0.25% trypsin for 1 h at 37°C. The cells were lysed by passage through a 1-ml syringe with a 25-gauge needle 40 times. Cell lysates were incubated for 1 h at 37°C once more, the trypsin was inactivated, and the samples were analyzed by Western blot analysis with a cocktail of HPV16 L1-specific mouse MAbs (IID5, 33L1-7, and 312F).

Article Snippet: The microtubule network was detected using mouse MAb anti-α-tubulin conjugated to Alexa Fluor 488 (AF488; catalog no. 8058S; Cell Signaling).

Techniques: Infection, Live Cell Imaging, Labeling, Incubation, Western Blot