Journal: The Journal of Biological Chemistry

Article Title: Lipopolysaccharide Primes the NALP3 Inflammasome by Inhibiting Its Ubiquitination and Degradation Mediated by the SCF FBXL2 E3 Ligase *

doi: 10.1074/jbc.M115.645549

Figure Lengend Snippet: FBXL2 triggers NALP3 degradation. A, MLE cells were transfected with increasing amounts of FBXL2 plasmid for 24–48 h. Cells were collected and assayed for V5 (FBXL2), NALP3, and β-actin by immunoblotting. B, MLE cells were transfected with several FBXL2 lentiviral shRNAs (4 μg). Human primary macrophages were also transfected with several siRNAs to FBXL2 (500 nm) for gene silencing. Shown are protein levels of FBXL2 after transfection of shRNAs or siRNAs and corresponding levels of NALP3 protein. Compared with a control RNA, despite a modest reduction in FBXL2 levels, increased NALP3 levels in the shRNA or siRNA groups were observed. C, U937 cells were lysed, and FBXL2 proteins were coimmunoprecipitated (IP) followed by NALP3, FBXL2, or NALP6 immunoblotting (IB, top panel). In addition, Beas2B cells were transfected with NALP3-V5 prior to IP for V5 and then, NALP3 and various F box proteins were immunoblotted (bottom blot). IgG was used as a control in all IP experiments, and inputs are the IBs of 10% of the cell lysates before performing IP. Note that an upper IgG chain band was detected in the FBXL7 immunoblot. D, in vitro ubiquitination assays showing that NALP3 is a substrate for FBXL2-mediated ubiquitination. E, A549 cells were incubated with or without LPS (200 ng/ml) for 16 h, and cells were lysed. FBXL2 protein was coimmunoprecipitated; first IP for FBXL2 followed by NALP3, FBXL2, or β-actin immunoblotting. F, the relative association between FBXL2 and NALP3 after LPS was quantified using densitometric analysis of the bands in E. The binding capacity between NALP3 and FBXL2 is reduced by ∼45–50% after LPS exposure. Data are representative of at least two to three independent experiments.

Article Snippet: Human primary alveolar macrophages from the fluid were cultured in culture medium (Celprogen) as described previously ( 9 ).

Techniques: Transfection, Plasmid Preparation, Western Blot, shRNA, In Vitro, Incubation, Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: Lipopolysaccharide Primes the NALP3 Inflammasome by Inhibiting Its Ubiquitination and Degradation Mediated by the SCF FBXL2 E3 Ligase *

doi: 10.1074/jbc.M115.645549

Figure Lengend Snippet: An FBXO3 inhibitor reduces NALP3 abundance, thereby decreasing cytokine release. A, U937 cells were treated with BC-1215 at different concentrations for 16 h. Cells were collected for FBXL2 and β-actin immunoblotting. B, U937 and THP1 cells (3 × 106 cells each) were incubated with LPS (200 ng/ml) or BC-1215 (8 μg/ml) for 16 h. Cells were collected and lysed for NALP3, NALP6 (negative control), and GAPDH/β-actin immunoblotting. C and D, U937 cells (3 × 106 cells) were primed with LPS (200 ng/ml) in combination with different concentrations of BC-1215 for 16 h as indicated (C) or primed with LPS (200 ng/ml for 16 h) and then exposed to BC-1215 (4 μg/ml) in fresh culture medium for the indicated periods of time (D). Cells were collected to measure NALP3 and β-actin protein (top panels) and mRNA levels (bottom panels). Box plots of the -fold increase of steady-state NALP3 mRNA are shown. Data represent four independent experiments. The p values were determined by a Kruskal-Wallis test. E, U937 cells were incubated in LPS (200 ng/ml) with or without BC-1215 (4 μg/ml for 16 h). Cells were then exposed to CHX (40 μg/ml) at different time points for a half-life study. Immunoblotting for NALP3 and GAPDH (loading control) was performed. Densitometric plots of adjusted NALP3 protein decay over time under different conditions were best fitted. The half-life of LPS-primed NALP3 protein was reduced with BC-1215 treatment comparable with native conditions. Data are mean ± S.E. of two independent experiments. F, MLE cells were transfected either with WT or point mutant NALP3 plasmids for 48 h. LPS (40 μg/ml) was then added to the medium with or without BC-1215 (20 μg/ml) for 18 h. Cells were collected and lysed for NALP3 (V5-tagged) and β-actin (loading control) immunoblotting. Levels of K689R, the point mutant for a putative ubiquitin acceptor site within the NALP3 protein, did not decrease with BC-1215 compared with WT or mutant NALP3. Data are representative of two independent experiments. G, THP1 and K562 cells (3 × 106 cells each) were incubated with LPS (200 ng/ml) with or without BC-1215 (8 μg/ml) for 20 h and then pulsed with ATP (5 mm for 20 min). Culture medium was collected for immunoblotting of pro-IL-1β, IL-1β, pro-IL-18, and IL-18. H, THP1 cells (3 × 106 cells each) were incubated with LPS (200 ng/ml) for 20 h and different time periods of BC-1215 (8 μg/ml), as indicated, in the same volume of culture medium. Cells were then exposed to ATP (5 mm for 20 min). Culture medium was collected for immunoblotting of pro-IL-1β and IL-1β. The ratio of IL-1β versus pro-IL-1β by densitometry is shown (bottom panel). Data represent mean ± S.D. of two independent experiments. The p value was determined by nonparametric test for trend. I, primary human alveolar macrophages were obtained from a healthy volunteer. Human alveolar macrophages (3 × 105 cells) were incubated with BC-1215 (4 μg/ml) for 2 h and then exposed to LPS (100 ng/ml) for 16 h. Cells were lysed and collected for immunoblotting for NALP3 and β actin. J, primary human alveolar macrophages (3 × 105 cells) from the same subject as in I were incubated with BC-1215 (4 μg/ml) for 2 h and then exposed to LPS (100 ng/ml) for 16 h. ATP (5 mm) was added for 15 min before collecting cell culture supernatants for ELISA. Data represent the mean ± S.D. of duplicate measurements.

Article Snippet: Human primary alveolar macrophages from the fluid were cultured in culture medium (Celprogen) as described previously ( 9 ).

Techniques: Western Blot, Incubation, Negative Control, Transfection, Mutagenesis, Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: Bioactive Materials

Article Title: Advances in the engineering of the outer blood-retina barrier: From in-vitro modelling to cellular therapy

doi: 10.1016/j.bioactmat.2023.08.003

Figure Lengend Snippet: From simple to complex oBRB co-culture models. 1/ ARPE-19 and HUVECs cultured on the two sides of a decellularized amniotic membrane. (A) Toluidine blue-stained trilayer showing monolayers of the RPE (above) and the HUVECs (below). (B) Confocal micrographs of optical sections of the trilayer tilted around its axis, with images have been tilted at varying angles [starting with the RPE surface (left side) to the HUVEC surface (right side)] using Velocity software and with immunostaining for ZO-1 (green) and occludin (red). Adapted from Hamilton et al. with permission of John Wiley and Sons. 2/ PDMS-based chip device for the 3D co-culture of an ARPE-19 monolayer above a microvessel cultured with HUVECs. (A) Schematic representation of the patterning of a collagen I hydrogel using the subtractive method to create the microvessel. (B) (i) 3D construction of confocal image of the co-culture system stained for nuclei and actin filaments, revealing (ii) an equal distribution of HUVECs along the microchannel as well as (iii) the ARPE-19 monolayer located above the microvessel. These cells were positive for their respective cell–cell adhesion markers: (iv) VE-cadherin for HUVEC and (v) ZO-1 for ARPE-19. Scale bars: 50 μm. Adapted with permission (Creative Commons Attribution 3.0 Unported Licence) from Arik et al. 3/ Microengineered model of the oBRB with complex vascularization. (A) Schematic view of the oBRB and (B) its modelization by culturing human iPSC-derived RPEs on the surface of a hydrogel construct containing a network of blood vessels. (C) Microvessels at day 14 constructed by primary human retinal microvascular endothelial in co-culture with choroidal fibroblasts embedded in a fibrin scaffold. (D) Well-defined intercellular tight junctions (ZO-1) in the iPSC-RPE monolayer after 14 days of culture. The beneficial effects of the co-culture of RPE cells with ECs, compared to a RPE monoculture are observed in (E–F) the production of basement membrane proteins by the RPE on the basal side after 14 days and in (G–H) the increased expression of RPE-specific markers such as RPE65 after 14 days. Scale bars are 50 μm ***P < 0.001 (n = 3). Adapted with permission from Paek et al. . Copyright from American Chemical Society. Figure created with BioRender.com .

Article Snippet: A commercial human choroidal endothelial primary cell line that can be maintained in culture for up to 12 passages is currently available (Celprogen Inc.), but, to our knowledge, it has not yet been used for development of BRB model.

Techniques: Co-Culture Assay, Cell Culture, Membrane, Staining, Software, Immunostaining, Derivative Assay, Construct, Expressing

Journal: Bioactive Materials

Article Title: Advances in the engineering of the outer blood-retina barrier: From in-vitro modelling to cellular therapy

doi: 10.1016/j.bioactmat.2023.08.003

Figure Lengend Snippet: oBRB disease modelling. 1/ Co-culture model of the choroidal neovascularization (CNV) in wet-AMD. (A) Scheme of the RPE–choroid complex underlying the neural retina of the eye and design of the microfluidic device to mimic it, using HUVECs, ARPE-19 and fibroblasts. (B) Schematic illustration of CNV, with the RPE layer and Bruch's membrane being destroyed by angiogenic sprouting of the choroid induced by an abnormal VEGF (vascular endothelial growth factor) gradient. (C) Representative images of VEGF treatment with or without bevacizumab. Excessive angiogenic sprouts in the gap channels were regarded as neo vessel formation. Cotreatment of VEGF with bevacizumab inhibited angiogenic sprouting. Scale bar: 200 μm. (D) Measurements of the area of angiogenic sprouting in the gap channel. The boundary of the gap and choroidal channel is indicated as a dotted line in (C). *P < 0.05. n = 3 for each condition. Adapted from Chung et al. with permission of John Wiley and Sons. 2/ Mono-culture model of SFD and DHRD. Formation of sub-RPE deposits with drusen-like composition underneath aged (D90) SFD and DHRD hiPSC-RPE cultures. (A–D) Confocal images of cellular cross-sections displayed the presence of TIMP3-APOE–positive (A), EFEMP1-APOE–positive (B), CRYAA/CRYAB-APOE–positive (C), and APOE-positive deposits underlying basement membrane marked by COL4 (D) in SFD and DHRD hiPSC-RPE cultures. Scale bar: 25 μm. (E–F) Quantitative Western blot analyses revealed increased amount of COL4 protein in the ECM underlying SFD and DHRD hiPSC-RPE cultures compared with Ctrl hiPSC-RPE cultures at D90. Data are presented as mean + SEM. *P ≤ 0.05. Adapted with permission from Galloway et al. . 3/ Mono-culture model of oculocutaneous albinism. hiPSC-RPE from OCA1A and OCA2 patients, cultured on Transwell recapitulates the disease characteristic pigmentation defects, by demonstrating various amounts of melanin and melanosomes (A–B) Brightfield microscopy images and transmission electron microscopy of CTRL-, OCA1A-, and OCA2-hPSC-RPE monolayers. Scale bar: 200 μm and 2 μm respectively. ( C –D) Quantification of the number of melanosomes per cell and quantification of degenerating melanosomes in CTRL-iRPE (N = 32), OCA1A-iRPE (N = 20), and OCA2-iRPE (N = 20), where N is the number of unique TEM images represents pooled data from a single differentiation event of four different lines in each group. The horizontal lines in the box plots indicate the median, the boxes indicate the first and third quartiles, and the whiskers indicate the 5th and 95th percentiles. Student's t -test was used to determine p values. Adapted from Georges et al. with permission of Elsevier.

Article Snippet: A commercial human choroidal endothelial primary cell line that can be maintained in culture for up to 12 passages is currently available (Celprogen Inc.), but, to our knowledge, it has not yet been used for development of BRB model.

Techniques: Co-Culture Assay, Membrane, Western Blot, Cell Culture, Microscopy, Transmission Assay, Electron Microscopy

Journal: Tissue Engineering and Regenerative Medicine

Article Title: Potency of Human Urine-Derived Stem Cells for Renal Lineage Differentiation

doi: 10.1007/s13770-017-0081-y

Figure Lengend Snippet: Flow cytometric analysis of cell surface marker expression on mesenchymal stem cells. The values were normalized to the isotype IgG control. Expression of these markers was compared to the levels expressed at week 0 (in the undifferentiated phase before induction of renal-lineage differentiation). For urine-derived stem cells (USCs), 95% or more expressed the mesenchymal stem cell markers CD44 and CD73, whereas <3% expressed the hematopoietic lineage markers CD34 and CD45. Ctrl renal stem cells; ADSC adipose tissue-derived stem cells; AFSC amniotic fluid-derived stem cells; USC urine-derived stem cells

Article Snippet: Human renal stem cells (Cat. no. 36100-27; RSCs; Celprogen, Torrance, CA, USA) were used as an indicator cell line for renal lineage induction and were cultured in human kidney stem cell medium (Cat. no. M36100-27S; Celprogen).

Techniques: Marker, Expressing, Derivative Assay

Journal: Tissue Engineering and Regenerative Medicine

Article Title: Potency of Human Urine-Derived Stem Cells for Renal Lineage Differentiation

doi: 10.1007/s13770-017-0081-y

Figure Lengend Snippet: Characterization of in vitro renal lineage-differentiated cells by morphological, immunocytochemical (ICC), quantitative real-time PCR, and secreted trophic factors analysis for 3 weeks. A Representative images from the morphological analysis. The original cell morphology (spindle-like) of the urine-derived stem cells (USCs) gradually changed to a large, round phenotype with a cobble stone-like appearance and cell aggregation. Representative ICC images using SSEA4, Pax2, Wt1, and Cadherin-6 antibodies. B In USCs, the stem cell marker SSEA4 was strongly expressed in undifferentiated stem cells, and then diminished in the differentiated phase. C Pax2 showed widespread nuclear expression at week 0, and then expression gradually became more localized and increased over time, and it was highly expressed in the cell aggregates at week 3. D Wt1 expression in the cytosol gradually increased over time. E Cadherin-6 was expressed in the cytoplasm of a few cells. The target proteins are shown in red, and the nucleus was stained with 4,6–diamidino-2–phenylindole (DAPI, blue). F Real-time PCR analysis. USCs showed the highest expression of LIM1, CD24, and OCLN. G Secreted trophic factor analysis by ELISA. VEGF and PDGF-bb were more strongly expressed in USCs than in ADSCs and AFSCs. The different letters on top of the bars indicate significant differences at p < 0.05. Ctrl renal stem cells, ADSC adipose tissue-derived stem cells, AFSC amniotic fluid-derived stem cells, USC urine-derived stem cells. (Color figure online)

Article Snippet: Human renal stem cells (Cat. no. 36100-27; RSCs; Celprogen, Torrance, CA, USA) were used as an indicator cell line for renal lineage induction and were cultured in human kidney stem cell medium (Cat. no. M36100-27S; Celprogen).

Techniques: In Vitro, Real-time Polymerase Chain Reaction, Derivative Assay, Marker, Expressing, Staining, Enzyme-linked Immunosorbent Assay

Journal: Tissue Engineering and Regenerative Medicine

Article Title: Potency of Human Urine-Derived Stem Cells for Renal Lineage Differentiation

doi: 10.1007/s13770-017-0081-y

Figure Lengend Snippet: In vivo safety analysis of renal-differentiated cells. Renal lineage-differentiated USCs were implanted into the subcapsule of the kidney. Four weeks later, a histological analysis was performed. No abnormal morphology was observed in the implanted kidneys. Ctrl renal stem cells, ADSC adipose tissue-derived stem cells, AFSC amniotic fluid-derived stem cells, USC urine-derived stem cells

Article Snippet: Human renal stem cells (Cat. no. 36100-27; RSCs; Celprogen, Torrance, CA, USA) were used as an indicator cell line for renal lineage induction and were cultured in human kidney stem cell medium (Cat. no. M36100-27S; Celprogen).

Techniques: In Vivo, Derivative Assay