Journal: bioRxiv

Article Title: Virological characteristics of SARS-CoV-2 BA.2 variant

doi: 10.1101/2022.02.14.480335

Figure Lengend Snippet: a,h , Growth kinetics of SARS-CoV-2 variants. b , Fluorescence microscopy. GFP area of infected VeroE6/TMPRSS2 cells at 48 h.p.i were measured. Numbers in the panel indicate the number of GFP-positive cells counted. c , Plaque assay. Diameter of plaques (20 plaques per virus) are summarized. d,f , S-based fusion assay. The fusion activity (arbitrary units) is shown. e , Western blot. Left, representative blots of S-expressing cells. ACTB is an internal control. Right, the ratio of S2 to the full-length S plus S2 proteins. g , Fold increase of pseudovirus infectivity by TMPRSS2 expression. Assays were performed in quadruplicate ( a,g,h ), octuplicate ( a , most left) or triplicate ( d–f ) and data are the average ± s.d. Each dot indicates the result from an individual plaque ( c ) and an individual replicate ( e,g ). In b and c , raw data are shown in and . Statistically significant differences between BA.2 and other variants through timepoints were determined by multiple regression ( a,d,f,h ). Familywise error rates (FWERs) calculated using the Holm method are indicated in the figures. Statistically significant differences between BA.1 and BA.2 were determined by two-sided Mann–Whitney U -tests ( b,d ) or two-sided paired Student’s t -tests ( e ).

Article Snippet: To measure the surface expression level of TMPRSS2 , HEK293-ACE2 cells and HEK293-ACE2/TMPRSS2 cells were stained with rabbit anti-TMPRSS2 polyclonal antibody (BIOSS, Cat# BS-6285R, 1:100).

Techniques: Fluorescence, Microscopy, Infection, Plaque Assay, Single Vesicle Fusion Assay, Activity Assay, Western Blot, Expressing, MANN-WHITNEY

Journal: bioRxiv

Article Title: Virological characteristics of SARS-CoV-2 BA.2 variant

doi: 10.1101/2022.02.14.480335

Figure Lengend Snippet: a, Fluorescence microscopy. GFP area of infected VeroE6/TMPRSS2 cells (m.o.i. 0.01) at 24, 48, and 72 h.p.i were measured. Higher-magnification views of the regions indicated by squares are shown at bottom. b , Plaque assay. c , Coculture of S-expressing cells with HEK293-ACE2/TMPRSS2 cells. Left, representative images of S-expressing cells cocultured with HEK293 cells (top) or HEK293-ACE2/TMPRSS2 cells (bottom). Nuclei were stained with Hoechst 33342 (blue). Right, the size distribution of syncytia (green). Numbers in the panel indicate the numbers of GFP-positive syncytia counted. Data are the average ± s.d. A statistically significant difference between BA.1 an BA.2 was determined by two-sided Mann–Whitney U -test. In a and b , summarized data are shown in and . Scale bars, 500 μm ( a ) or 200 μm ( c ).

Article Snippet: To measure the surface expression level of TMPRSS2 , HEK293-ACE2 cells and HEK293-ACE2/TMPRSS2 cells were stained with rabbit anti-TMPRSS2 polyclonal antibody (BIOSS, Cat# BS-6285R, 1:100).

Techniques: Fluorescence, Microscopy, Infection, Plaque Assay, Expressing, Staining, MANN-WHITNEY

Journal: bioRxiv

Article Title: Virological characteristics of SARS-CoV-2 BA.2 variant

doi: 10.1101/2022.02.14.480335

Figure Lengend Snippet: a , S expression on the cell surface. Representative histograms stained with anti-S1/S2 polyclonal antibody (left) and the summarised data (right) are respectively shown. The number in the histogram indicates MFI. Grey histograms indicate isotype controls. b , Binding affinity of SARS-CoV-2 S RBD to ACE2 by yeast surface display. Left, The percentage of the binding of the SARS-CoV-2 S RBD expressed on yeast to soluble ACE2 (left) and the summarised data (right) are respectively shown. c , TMPRSS2 expression on the cell surface. Left, representative histograms stained with anti-TMPRSS2 polyclonal antibody are shown. The number in the histogram indicates MFI. Grey histograms indicate isotype controls. Right, summarized data. Assays were performed in triplicate, and data are the average ± s.d. Each dot indicates the result from an individual replicate. A statistically significant difference between BA.1 an BA.2 was determined by two-sided unpaired Student’s t -test ( a ) or two-sided Mann–Whitney U -test ( b ).

Article Snippet: To measure the surface expression level of TMPRSS2 , HEK293-ACE2 cells and HEK293-ACE2/TMPRSS2 cells were stained with rabbit anti-TMPRSS2 polyclonal antibody (BIOSS, Cat# BS-6285R, 1:100).

Techniques: Expressing, Staining, Binding Assay, MANN-WHITNEY

Journal: Arthritis Research & Therapy

Article Title: Connective tissue growth factor promotes articular damage by increased osteoclastogenesis in patients with rheumatoid arthritis

doi: 10.1186/ar2863

Figure Lengend Snippet: Connective tissue growth factor expression in synovial tissue of patients with rheumatoid arthritis. The representative results of HE staining (Figure 2A), immunofluorescence anti-CTGF antibody staining (Figure 2B; green), and anti-F4/80 antibody staining (Figure 2C; red) are shown using surgical samples from RA and OA patients. The samples were counterstained by DAPI (blue) for nuclear staining and merge images are shown (Figure 2D). A strong expression of CTGF and F4/80 was observed in the samples of RA compared to OA and the CTGF expression cells were not overlapped with F/40 expression cells indicating that CTGF is upregulated in synovial fibroblasts rather than macrophages.

Article Snippet: The serum level of CTGF in human sera was evaluated by a sandwich ELISA system using two different anti-human CTGF antibodies; monoclonal anti-human CTGF antibody (R&D System, Cat#MAB660) and biotinated anti-human CTGF antibody (R&D System, Cat#BAF660).

Techniques: Expressing, Staining, Immunofluorescence

Journal: Arthritis Research & Therapy

Article Title: Connective tissue growth factor promotes articular damage by increased osteoclastogenesis in patients with rheumatoid arthritis

doi: 10.1186/ar2863

Figure Lengend Snippet: Synergistic effects of connective tissue growth factor on M-CSF/sRANKL-mediated osteoclastic function. Figure 5A shows the results of the resorption of osteoclasts on calcium phosphate. Vacant regions indicated by arrows represent the areas where the osteoclasts actually absorbed. There was no vacant region in negative control cells (M-CSF alone). In contrast to negative control, significant vacant regions were observed in osteoclasts induced by M-CSF/sRANKL. CTGF further expanded the vacant areas in combination with M-CSF/sRANKL and anti-CTGF antibody neutralized this effect. Figure 5B shows the levels of expression of osteoclasts specific proteases (MMP-9 and cathepsin-K) measured by quantitative real time RT-PCR. Synergistic effect of CTGF was also observed for M-CSF/sRANKL-mediated osteoclastogenesis. Bars in Figure 5B indicate the SD.

Article Snippet: The serum level of CTGF in human sera was evaluated by a sandwich ELISA system using two different anti-human CTGF antibodies; monoclonal anti-human CTGF antibody (R&D System, Cat#MAB660) and biotinated anti-human CTGF antibody (R&D System, Cat#BAF660).

Techniques: Negative Control, Expressing, Quantitative RT-PCR

Journal: Arthritis Research & Therapy

Article Title: Connective tissue growth factor promotes articular damage by increased osteoclastogenesis in patients with rheumatoid arthritis

doi: 10.1186/ar2863

Figure Lengend Snippet: Connective tissue growth factor mediate ERK1/2 and focal adhesion kinase activation through integrin αVβ3 signal transduction. Figure 6A shows the immnoprecipitation and immunoblotting analysis. The cell extracts of osteoclasts stimulated with recombinant CTGF (10 or 50 ng/ml) at 60 minutes were precipitated using anti-integrin αVβ3 antibody and subsequently blotted with anti- phosphorylated ERK1/ERK2, conventional ERK1/ERK2, and integrin αVβ3 antibodies respectively. The phosphorylated ERK1/ERK2 was recruited with integrin αVβ3 by CTGF stimulation. Figure 6B shows the immunoblotting analysis using anti- phosphorylated FAK, conventional FAK, and β-actin antibodies in the osteoclasts extracts treated with CTGF (10 ng/ml) at 5, 15, 60, and 120 minutes in the presence or absence of anti-CTGF antibody (1 μg/ml). CTGF stimulation resulted in phosphorylation of FAK from 60 minutes and this effect was neutralized by anti-CTGF antibody suggesting activation of signal transduction pathways through integrin αVβ3.

Article Snippet: The serum level of CTGF in human sera was evaluated by a sandwich ELISA system using two different anti-human CTGF antibodies; monoclonal anti-human CTGF antibody (R&D System, Cat#MAB660) and biotinated anti-human CTGF antibody (R&D System, Cat#BAF660).

Techniques: Activation Assay, Transduction, Western Blot, Recombinant, Phospho-proteomics

Journal: Nature Communications

Article Title: RNF168 and USP10 regulate topoisomerase IIα function via opposing effects on its ubiquitylation

doi: 10.1038/ncomms12638

Figure Lengend Snippet: ( a ) Identification of RNF168-associated proteins. A representative SDS–polyacrylamide gel electrophoresis of Flag-RNF168-associated proteins. Flag-tagged RNF168 was transfected in HEK293T cells and pull-down analysis was performed 48 h later. Protein bands were detected by silver staining. Protein bands were identified by mass spectrometry analysis following in-gel protease digestion. ( b ) HEK293T cells were transfected as indicated with HA-tagged RNF168 and Flag-TOP2α expression vectors. Cells were lysed and IP was performed using anti-Flag antibody. The resulting precipitates were subjected to IB analysis with the indicated antibodies. WCL, whole-cell lysate. ( c ) TOP2α, RNF168 and IgG (control) immunoprecipitates from HEK293T cells were examined by IB as indicated. ( b , c ) Data are representative of three independent experiments. ( d ) Cells treated with EdU were used for detection of localization patterns of TOP2α (Alexa Fluor 488) and RNF168 (Alexa Fluor 594) using confocal microscopy. Scale bar, 20 μm.

Article Snippet: After washing with 3% BSA in PBS, the sheep polyclonal against RNF168 (1:2,000; R&D systems cat# AF7217) and the rabbit polyclonal against TOP2α (Abcam) were detected using their respective secondary antibodies.

Techniques: Polyacrylamide Gel Electrophoresis, Transfection, Silver Staining, Mass Spectrometry, Expressing, Control, Confocal Microscopy

Journal: Nature Communications

Article Title: RNF168 and USP10 regulate topoisomerase IIα function via opposing effects on its ubiquitylation

doi: 10.1038/ncomms12638

Figure Lengend Snippet: ( a ) Representative agarose gel of in vitro kinetoplast DNA-based decatenation assays performed for 10 min with different amount of nuclear extracts from WT and Rnf168 −/− MEFs. Catenated and decatenated kDNA were separated by electrophoresis using 1% agarose gel. IB show Top2α's level in the total nuclear extracts used for this assay. ( b ) Representative data of the mitotic inhibition assay of decatenation G2 checkpoint in WT and Rnf168 −/− primary MEFs. Cells were treated with DMSO or ICRF-193 for 15 min and then incubated in culture media for an additional 2 h. The fraction of mitotic cells (pHH3 + ) was determined by flow cytometry. ( c ) Bar graphs represent the mean inhibition of mitotic index 2 h post ICRF-193 treatment of passage 1 primary MEFs (% pHH3 + cells post ICRF-193 treatment compared with DMSO-treated controls). * P <0.05. ( d ) Bar graphs represent the mean inhibition of mitotic index 2 h post ICRF-193 treatment of RIDDLE cells reconstituted with either HA-RNF168 or HA-empty vector as in c . * P <0.05. ( e ) Analysis of decatenation G2 checkpoint of Rnf168 −/− and WT 3T3 MEFs using the mitotic entry assay. Percentage of pHH3 + cells is shown at the indicated time post-treatment with colcemid in the presence of DMSO or ICRF-193. * P <0.05; Rnf168 −/− MEFs compared with WT MEFs 6 h post-ICRF-193 treatment. ( f ) Bar graphs represent the mean fraction of pHH3 + WT and Rnf168 −/− MEFs evading G2 arrest 6 h post-treatment with ICRF-193 compared with DMSO-treated cells as in e . * P <0.05. ( g ) Mitotic entry assay of decatenation G2 checkpoint in human RIDDLE cells reconstituted with HA-empty vector or HA-RNF168. Percentage of pHH3 + cells is shown at the indicated times post-treatment with colcemid with or without ICRF-193. * P <0.05, RIDDLE cells reconstituted with HA-empty vector compared with RIDDLE cells reconstituted with HA-RNF168 at 6 h post-ICRF-193 treatment. ( h ) Bar graphs represent the mean fraction of RIDDLE cells (reconstituted with HA-empty vector compared with those reconstituted with HA-RNF168) evading G2 arrest 6 h post ICRF-193 treatment compared with DMSO-treated controls as in f . * P <0.05. Three independent experiments in triplicates unless specified. Error bars in c , d , f and h represent mean±s.e.m.

Article Snippet: After washing with 3% BSA in PBS, the sheep polyclonal against RNF168 (1:2,000; R&D systems cat# AF7217) and the rabbit polyclonal against TOP2α (Abcam) were detected using their respective secondary antibodies.

Techniques: Agarose Gel Electrophoresis, In Vitro, Electrophoresis, Inhibition, Incubation, Flow Cytometry, Plasmid Preparation

Journal: Nature Communications

Article Title: RNF168 and USP10 regulate topoisomerase IIα function via opposing effects on its ubiquitylation

doi: 10.1038/ncomms12638

Figure Lengend Snippet: ( a – d ) Sensitivity of WT and Rnf168 −/− MEFs to ICRF-193 ( a , b ) or etoposide ( c , d ) was determined using clonogenic assays. ( e , f ) Sensitivity of the human breast cancer cell lines T47D and MDA-MB-231 to etoposide was determined using clonogenic assays. ( a , c , e ) Data are presented as the mean±s.e.m. ( a , c , n >4; e , n =4). * P <0.05 for Rnf168 −/− MEFs compared with WT MEFs and T47D and MDA-MB-231 cells knocked down for RNF168 (sh.RNF168) compared with their respective controls (Sh.Ctr: ShRNA control). ( b , d ) Representative pictures of dishes showing surviving WT and Rnf168 −/− colonies post ICRF-193 ( b ) or etoposide ( d ) treatment. ( f ) Representative pictures of dishes showing surviving colonies of etoposide treated T47D and MDA-MB-231 cells.

Article Snippet: After washing with 3% BSA in PBS, the sheep polyclonal against RNF168 (1:2,000; R&D systems cat# AF7217) and the rabbit polyclonal against TOP2α (Abcam) were detected using their respective secondary antibodies.

Techniques: shRNA, Control

Journal: Nature Communications

Article Title: RNF168 and USP10 regulate topoisomerase IIα function via opposing effects on its ubiquitylation

doi: 10.1038/ncomms12638

Figure Lengend Snippet: ( a ) RIDDLE cells reconstituted with HA-RNF168 or HA-empty vector, and control HA-RNF168-reconstituted RIDDLE cells with TOP2α knock down were lysed and WCL subjected to IP with anti-TOP2α or IgG (control) antibodies. Immunoprecipitates were blotted with the indicated antibodies to detect ubiquitylated TOP2α. ( b ) Human breast cancer cell lines T47D and MDA-MB-231 knocked down for RNF168 (Sh.RNF168) and their control expressing ShRNA control (sh.Ctr) were examined for their level of ubiquitylated TOP2α as in a . ( c ) Rnf168 −/− , Brca1 −/− and WT MEFs were lysed and subjected to IP with anti-Top2α or IgG (control) antibodies. IPs from WCL were blotted with the indicated antibodies. ( d ) HEK293T cells were transfected with RNF168 (WT or mutant Rnf168-C21S), Flag-TOP2α and HA-Ub vectors as indicated. WCL were subjected to IP with anti-Flag, and IB analysis was performed using anti-HA antibody to detect ubiquitylated Flag-TOP2α. ( e ) Nuclear extracts from RIDDLE cells reconstituted with HA-RNF168 or HA-empty vector were subjected to IP with anti-TOP2α or IgG (control) antibodies. Immunoprecipitates were blotted with the indicated antibodies against K63- and K48-Ub linkages. ( f ) In vitro ubiquitylation of recombinant TOP2α in the presence of recombinant RNF168 (500 ng for lane 5, 1 μg for lanes 2, 3 and 6 and 2 μg for lane7), UBE1 (E1), UBE2E2 (E2) and Ub. Nuc, nuclear extract; WCL, whole-cell lysate.

Article Snippet: After washing with 3% BSA in PBS, the sheep polyclonal against RNF168 (1:2,000; R&D systems cat# AF7217) and the rabbit polyclonal against TOP2α (Abcam) were detected using their respective secondary antibodies.

Techniques: Plasmid Preparation, Control, Knockdown, Expressing, shRNA, Transfection, Mutagenesis, In Vitro, Recombinant

Journal: Nature Communications

Article Title: RNF168 and USP10 regulate topoisomerase IIα function via opposing effects on its ubiquitylation

doi: 10.1038/ncomms12638

Figure Lengend Snippet: ( a ) WT and Rnf168 −/− MEFs, and Top2α knockdown control MEFs were fixed and stained with DAPI. Representative cells with defective chromosome segregation, as indicated by chromosome bridges (arrow head) and micronuclei (arrow) are shown. ( b ) Histograms show quantification of cells with chromosome bridges or micronuclei (mean±s.e.m., n =3). * P <0.05. ( c ) Histograms show the fraction of RIDDLE cells reconstituted with HA-RNF168 or HA-empty vector that display micronuclei (mean±s.e.m., n =3). * P <0.05. ( d ; upper panels) Representative metaphase spreads showing undercondensed chromosomes in WT MEFs 24 h post ICRF-193 treatment (positive control), and in Rnf168 −/− DMSO-treated MEFs. ( d , lower panels) Representative metaphase spreads of DMSO-treated Rnf168 −/− MEFs complemented with RNF168-WT (WT) or the E3 ligase deficient RNF168-C21S (C21S). ( e ) Histograms show quantification of abnormal metaphase spreads with entangled or undercondensed chromosomes from the indicated cells (mean±s.e.m., n =3). * P <0.05 compared with WT MEFs. ( f ) Histograms present the mean fraction of MEFs evading ICRF-193-induced G2 arrest as compared with DMSO-treated controls (mean±s.e.m., n =3). Data are shown for WT MEFs, mock infected Rnf168 −/− MEFs and Rnf168 −/− MEFs complemented with RNF168-WT or RNF168-C21S. 3T3 MEFs were used for these experiments. Data shown are for 6 h post-treatment with colcemid±4 μM ICRF-193. * P <0.05 compared with WT MEFs. ( g ) Clonogenic assay was used to determine sensitivity to ICRF-193 of mock infected WT and Rnf168 −/− MEFs, as well as Rnf168 −/− MEFs complemented with RNF168 (WT or C21S mutant). Data are presented as the mean±s.e.m. ( n =4). * P <0.05 compared with WT MEFs. Scale bar, 20 μm.

Article Snippet: After washing with 3% BSA in PBS, the sheep polyclonal against RNF168 (1:2,000; R&D systems cat# AF7217) and the rabbit polyclonal against TOP2α (Abcam) were detected using their respective secondary antibodies.

Techniques: Knockdown, Control, Staining, Plasmid Preparation, Positive Control, Infection, Clonogenic Assay, Mutagenesis

Journal: Nature Communications

Article Title: RNF168 and USP10 regulate topoisomerase IIα function via opposing effects on its ubiquitylation

doi: 10.1038/ncomms12638

Figure Lengend Snippet: ( a ) Nuclear (Nuc) and chromatin (Chr) fractions prepared from WT, Rnf168 −/− and Brca1 −/− MEFs were analysed by IB for chromatin occupancy of Top2α. Additional IBs were performed with the indicated antibodies as controls. H4, histone H4. ( b ) Nuclear and chromatin fractions were prepared from the human breast cancer cell lines T47D and MDA-MB-231 knocked down for RNF168 (Sh.RNF168) and their controls (Sh.Ctr) and analysed by IB for the chromatin occupancy of TOP2α as in a . ( c ) HEK293T were transfected with Flag-TOP2α along with RNF168 (+) or empty vector (−) and their nuclear and chromatin fractions were prepared and examined by IB using the indicated antibodies. ( d ) A representative agarose gel showing decatenation activity of soluble nuclear and chromatin extracts from 2 WT and 2 Rnf168 −/− MEFs. In vitro kinetoplast DNA-based decatenation assay was performed for 20 min with different amounts of nuclear and chromatin extracts, and catenated and decatenated kDNA were separated by electrophoresis. IB using anti-Top2α was performed to show the level of Top2α present in each sample.

Article Snippet: After washing with 3% BSA in PBS, the sheep polyclonal against RNF168 (1:2,000; R&D systems cat# AF7217) and the rabbit polyclonal against TOP2α (Abcam) were detected using their respective secondary antibodies.

Techniques: Transfection, Plasmid Preparation, Agarose Gel Electrophoresis, Activity Assay, In Vitro, Electrophoresis

Journal: Nature Communications

Article Title: RNF168 and USP10 regulate topoisomerase IIα function via opposing effects on its ubiquitylation

doi: 10.1038/ncomms12638

Figure Lengend Snippet: ( a , b ) HEK293T cells were transfected with Flag-RNF168 and HA-USP10 vectors ( a ) or Flag-TOP2α and HA-USP10 vectors ( b ) as indicated. Cells were lysed and IP was performed using anti-Flag and anti-HA antibodies. The resulting precipitates were subjected to IB analysis with the indicated antibodies. WCL, whole-cell lysate. ( c ) HEK293T cells were transfected with Flag-TOP2α, RNF168, HA-USP10 and Myc-Ub vectors, as indicated. IP using anti-Flag and WCL was subjected to anti-Ub IB analysis to detect TOP2α ubiquitylation. ( d ) WT MEFs with knockdown of Usp10 (Sh1 and Sh2) and WT controls were examined for the level of Top2α ubiquitylation. Top2α was immunoprecipitated from whole-cell extracts and examined by IB for its ubiquitylation level using anti-Ub. IP using IgG was used as a control. The indicated antibodies were used for IB. ( e ) HEK293T cells were transfected with Flag-TOP2α with or without RNF168, HA-Ub, USP10 and USP10-C424A as indicated. TOP2α chromatin occupancy in these cells was examined by IB using anti-Flag antibodies and chromatin fractions (Chr). IB analysis of the chromatin fractions is also shown for the indicated antibodies. ( f ) A simplified model of RNF168-mediated regulation of TOP2α ubiquitylation and decatenation function.

Article Snippet: After washing with 3% BSA in PBS, the sheep polyclonal against RNF168 (1:2,000; R&D systems cat# AF7217) and the rabbit polyclonal against TOP2α (Abcam) were detected using their respective secondary antibodies.

Techniques: Transfection, Knockdown, Immunoprecipitation, Control

Journal: Oncology Letters

Article Title: Scutellarein suppresses Aβ-induced memory impairment via inhibition of the NF-κB pathway in vivo and in vitro

doi: 10.3892/ol.2019.10274

Figure Lengend Snippet: Scue attenuates Alzheimer's disease-like pathology in rats. (A) Latency of crossings and (B) average number of crossings in the Morris water maze spatial probe test of memory. Hippocampal expression of (C) Aβ, (D) SOD, (E) MDA and (F) Ach. (G) Nissl staining in the hippocampus. Scale bar, 50 µm. The expression levels of (H) Bcl-2, Bax and cleaved-caspase-3 were detected by western blotting. Densitometry analyses were conducted using β-actin as an internal reference. Representative results from repeated experiments are presented and data are presented as the mean ± standard deviation. n=6. **P<0.01, ***P<0.001 vs. the control group; # P<0.05, ## P<0.01, ### P<0.001 vs. the Aβ group. ^ P<0.05, ^^ P<0.01 vs. the Scu group. Aβ, amyloid β; Scu, scutellarin; Scue, scutellarein; SOD, superoxide dismutase; MDA, malondialdehyde; Ach, acetylcholine; Bcl-2, B cell lymphoma-2; Bax, apoptosis regulator BAX.

Article Snippet: The membranes were incubated with primary antibodies against nuclear factor of κ-light polypeptide gene enhancer in B cells inhibitor α [IκBα; cat. no. YS-(kt)-0907, phospho (p)-IκBα (cat. no. YS-KT1551) (Shanghai Yansheng Biochemical Reagent Co., Ltd., Shanghai, China), NF-κB, cleaved caspase-3 (cat. no. bs-0081R), B cell lymphoma-2 (Bcl-2; cat. no. bs-20351R), apoptosis regulator BAX (Bax; cat. no. bs-4564R), β-actin (cat. no. bs-0061R) and lamin-A (cat. no. bs-1839R) (1:1,000; BIOSS) overnight at 4°C.

Techniques: Expressing, Staining, Western Blot, Standard Deviation

Journal: Journal of Translational Medicine

Article Title: Development of patient-derived xenograft models from a spontaneously immortal low-grade meningioma cell line, KCI-MENG1

doi: 10.1186/s12967-015-0596-8

Figure Lengend Snippet: Immunostaining of original tumor, low passage, and high passage KCI-MENG1 cells. The original patient-derived tumor ( top row ) showed moderate and patchy immunoreactivity for epithelial membrane antigen (EMA); strong and diffuse immunostaining for progesterone receptor (PR); and a Ki-67 proliferative index of 2–3%. There was also strong immunostaining for N-cadherin and vimentin. KCI-MENG1-LP cells ( middle row ) and KCI-MENG1-HP cells ( bottom row ) maintained expression of EMA, N-cadherin, and vimentin but had significantly reduced PR expression compared to the original tumor. Whereas Ki-67 labeling was found in only a small number of cells in the original tumor and low passage cells, it was positive in virtually all P84 cells. Scale bar 50 µm.

Article Snippet: Primary antibodies used targeted the following proteins: EMA (cat.#247M-94), PR (cat.#323R-14), Ki-67 (cat.#275R-14), vimentin (cat.#347R-14; all from CellMarque, Rocklin, CA, USA), and N-cadherin (cat.#NBP1-48309, Novus Biologicals, Littleton, CO, USA).

Techniques: Immunostaining, Derivative Assay, Membrane, Expressing, Labeling

Journal: Journal of Translational Medicine

Article Title: Development of patient-derived xenograft models from a spontaneously immortal low-grade meningioma cell line, KCI-MENG1

doi: 10.1186/s12967-015-0596-8

Figure Lengend Snippet: Immunostaining of original patient tumor, low and high passage KCI-MENG1 cells, and subcutaneous xenograft tumor. The original patient-derived tumor showed moderate immunoreactivity for E-cadherin which was maintained in all in vitro and in vivo models. Scale bar 50 µm.

Article Snippet: Primary antibodies used targeted the following proteins: EMA (cat.#247M-94), PR (cat.#323R-14), Ki-67 (cat.#275R-14), vimentin (cat.#347R-14; all from CellMarque, Rocklin, CA, USA), and N-cadherin (cat.#NBP1-48309, Novus Biologicals, Littleton, CO, USA).

Techniques: Immunostaining, Derivative Assay, In Vitro, In Vivo

Journal: Journal of Translational Medicine

Article Title: Development of patient-derived xenograft models from a spontaneously immortal low-grade meningioma cell line, KCI-MENG1

doi: 10.1186/s12967-015-0596-8

Figure Lengend Snippet: Meningioma cell lines reported in the literature

Article Snippet: Primary antibodies used targeted the following proteins: EMA (cat.#247M-94), PR (cat.#323R-14), Ki-67 (cat.#275R-14), vimentin (cat.#347R-14; all from CellMarque, Rocklin, CA, USA), and N-cadherin (cat.#NBP1-48309, Novus Biologicals, Littleton, CO, USA).

Techniques: Southern Blot, Injection, Expressing, TRAP Assay, Activity Assay, Real-time Polymerase Chain Reaction

Journal: Journal of Translational Medicine

Article Title: Development of patient-derived xenograft models from a spontaneously immortal low-grade meningioma cell line, KCI-MENG1

doi: 10.1186/s12967-015-0596-8

Figure Lengend Snippet: Human meningioma mouse xenograft model KCI-MENG1-LPSX generated with the spontaneously immortal cell line KCI-MENG1-LP. Tumors from immunocompromised SCID mice were dissected ( a ) and the derivative cell line KCI-MENG1-LPSX CL was generated. The H&E staining of the mouse tumor revealed a pattern of moderately cellular meningothelial cells similar to the original patient tumor ( b ). The KCI-MENG1-LPSX CL cells were composed of the round-shaped cells similar to the high passage parent cell line KCI-MENG1-HP ( c ). The EMA, PR, and N-cadherin IHC of the mouse tumor highly resembled the original patient-derived tumor ( d top row ). The vimentin- and Ki-67-stained cells in the mouse tumor tissue were markedly more abundant and more intensely stained than in the original tumor ( d top row ). KCI-MENG1-LPSX CL cells displayed the same patterns of immunostaining as the high passage parent cell line KCI-MENG1-HP, including the loss of PR staining ( d bottom row ). Scale bar 50 µm.

Article Snippet: Primary antibodies used targeted the following proteins: EMA (cat.#247M-94), PR (cat.#323R-14), Ki-67 (cat.#275R-14), vimentin (cat.#347R-14; all from CellMarque, Rocklin, CA, USA), and N-cadherin (cat.#NBP1-48309, Novus Biologicals, Littleton, CO, USA).

Techniques: Generated, Staining, Derivative Assay, Immunostaining

Journal: bioRxiv

Article Title: Cullin1 represses systematic inflammasome activation by binding and catalyzing NLRP3 ubiquitination

doi: 10.1101/289637

Figure Lengend Snippet: A HEK293T cells were transfected or co-transfected with pcDNA3.1(+)-NLRP3, pcDNA3.1(+)-pro-IL-1β, pcDNA3.1(+)-pro-caspase-1, and pcDNA3.1(+)-ASC, as indicated. B HEK293T cells were co-transfected with pcDNA3.1(+)-NLRP3, pcDNA3.1(+)-pro-IL-1β, pcDNA3.1(+)-pro-caspase-1, and pcDNA3.1(+)-ASC, along with pcDNA3.1(+)-3Flag-ATP1β3, pcDNA3.1(+)-3Flag-PGM1, or pcDNA3.1(+)-3Flag-Cullin1, as indicated. Secreted IL-1β in the supernatants were analyzed by ELISA. C Daigrams of Cullin1 and Cullin1ΔC (upper). HEK293T cells were co-transfected with pcDNA3.1(+)-NLRP3, pcDNA3.1(+)-pro-IL-1β, pcDNA3.1(+)-pro-caspase-1, and pcDNA3.1(+)-ASC along with pcDNA3.1(+)-3XFlag-Cullin1 or pcDNA3.1(+)-3XFlag-Cullin1ΔC, as indicated. Secreted IL-1β in the supernatants were analyzed by ELISA. D HEK293T cells were co-transfected with pcDNA3.1(+)-NLRP3, pcDNA3.1(+)-pro-IL-1β, pcDNA3.1(+)-pro-caspase-1, and pcDNA3.1(+)-ASC along with pcDNA3.1(+)-3XFlag-Cullin1 or pcDNA3.1(+)-3XFlag-Cullin1ΔC. E HEK293T cells were co-transfected with pCAGGS-HA-NLRP3 and pcDNA3.1(+)-3XFlag-Cullin1, pcDNA3.1(+)-3XFlag-Cullin2, or pcDNA3.1(+)-3XFlag-Cullin3, respectively. The cell lysates were immunoprecipitated with anti-HA antibody and then immunoblotted with indicated antibodies. F HEK293T cells were co-transfected with pcDNA3.1(+)-NLRP3, pcDNA3.1(+)-ASC, pcDNA3.1(+)-pro-caspase-1, and pcDNA3.1(+)-pro-IL-1β along with pcDNA3.1(+)-3XFlag-Cullin1, pcDNA3.1(+)-3XFlag-Cullin2, or pcDNA3.1(+)-3XFlag-Cullin3, respectively. G HEK293T cells were transduced with lentiviruses stably expressing shRNA (sh-NC) and shRNA against Cullin1 (sh-Cullin1#1 and sh-Cullin1#2) and selected with puromycin for 2 weeks. Cullin1 and GAPDH mRNAs were determined by qRT-PCR (upper) and Cullin1 and GAPDH proteins were detected Western blot analyses (low). H HEK293T cells stably expressing sh-NC or sh-Cullin1#2 were co-transfected with pcDNA3.1(+)-NLRP3, pcDNA3.1(+)-pro-IL-1β, pcDNA3.1(+)-pro-caspase-1, and pcDNA3.1(+)-ASC, along with pcDNA3.1(+)-3XFlag-Cullin1. Data information: In ( A , D , F , and H ), secreted IL-1β in the supernatants were analyzed by ELISA (upper). Matured IL-1β (p17) and matured Casp-1 (p20) in the cell lysates were determined by immunoblot analyses with indicated antibodies (low). Data information: In ( A–D , G–H ), data shown are means ± SEM, *P<0.05, **P<0.01, ***P<0.0001.

Article Snippet: Human IL-1β/IL-1F2 Quantikine ELISA Kit (Cat# DLB50) was purchased from R&D Systems (Minnesota, USA).

Techniques: Transfection, Enzyme-linked Immunosorbent Assay, Immunoprecipitation, Transduction, Stable Transfection, Expressing, shRNA, Quantitative RT-PCR, Western Blot

Journal: bioRxiv

Article Title: Cullin1 represses systematic inflammasome activation by binding and catalyzing NLRP3 ubiquitination

doi: 10.1101/289637

Figure Lengend Snippet: A PMA-differentiated THP-1 macrophages were infected with H3N2 for 24 h or treated with LPS and ATP for 30 min. The cell lysates were immunoblotted with indicated antibodies. B CUL1, Cyclin E1, and GAPDH proteins were examined by Western blot analysis in THP-1 cells stably expressing CUL1. C Macrophages stably expressing CUL1 were treated with ATP for 30 min. D Macrophages stably expressing CUL1ΔC were treated with ATP or infected with H3N2. Secreted IL-1β in the supernatants were analyzed by ELISA. E Macrophages stably expressing sh-Cullin1#2 were treated with LPS for 6 h, LPS for 6 h and ATP for 30 min, or LPS for 6 h and Nigericin for 1 h, respectively. Matured IL-1β (p17) in the cell lysates was determined by immunoblot analysis with indicated antibodies. F Macrophages stably expressing sh-Cullin1#2 were treated with ATP for 30 min. G Macrophages stably expressing sh-Cullin1#2 were treated with Nigericin for 1 h. H Macrophages stably expressing sh-Cullin1#2 were treated with Alum for 6 h I Macrophages stably expressing sh-Cullin1#2 were infected with IAV H3N2 for 24 h J Macrophages stably expressing sh-Cullin1#2 were treated with Poly(dA:dT)/LyoVec TM for 12 h. Data information: In ( C , F–J ), secreted IL-1β in the supernatants were analyzed by ELISA (upper). Matured IL-1β (p17) and matured Casp-1 (p20) in the cell lysates were determined by immunoblot analysis with indicated antibodies (low). Data information: In ( C , D , F–J ), data shown are means ± SEM, *P<0.05, **P<0.01, ***P<0.0001.

Article Snippet: Human IL-1β/IL-1F2 Quantikine ELISA Kit (Cat# DLB50) was purchased from R&D Systems (Minnesota, USA).

Techniques: Infection, Western Blot, Stable Transfection, Expressing, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: Cullin1 represses systematic inflammasome activation by binding and catalyzing NLRP3 ubiquitination

doi: 10.1101/289637

Figure Lengend Snippet: A HEK293T cells were transfected with pcDNA3.1(+)-3XFlag-Cullin1 and pCAGGS-HA-NLRP3, pCAGGS-HA-PYD, pCAGGS-HA-NBD, pCAGGS-HA-LLR, respectively. B HEK293T cells were transfected with pCAGGS-HA-Cullin1 and pcDNA3.1(+)-3XFlag-NLRP3, pcDNA3.1(+)-3XFlag-ASC, or pcDNA3.1(+)-3XFlag-pro-caspase-1, respectively. C HEK293T cells were transfected with pcDNA3.1(+)-NLRP3 and pcDNA3.1(+)-ASC along with pcDNA3.1(+)-3XFlag-Cullin1. D HEK293T cells were transfected with pCAGGS-HA-NLRP3 and pCAGGS-HA-ASC along with pcDNA3.1(+)-3XFlag-Cullin1 at different concentrations. E HEK293T cells were co-transfected with pcDNA3.1(+)-NLRP3 and/or pcDNA3.1(+)-ASC along with pcDNA3.1(+)-3XFlag-Cullin1, as indicated. F HEK293T cells were co-transfected with pcDNA3.1(+)-NLRP3, pcDNA3.1(+)-ASC, pcDNA3.1(+)-3XFlag-Cullin1 and then treated with 5 mM ATP for 30 min and 10 μM Nigericin for 1 h. G PMA-differentiated THP-1 macrophages were stimulated with ATP or Nigericin, and infected with H3N2. H PMA-differentiated THP-1 macrophages were stimulated with ATP or Nigericin. The cells were immunostained with anti-NLRP3 and anti-Cullin1 antibodies. The sub-cellular localizations of endogenous NLRP3 (green), endogenous Cullin1 (Red), and nucleus marker DAPI (blue) were analyzed under confocal microscopy. I PMA-differentiated THP-1 macrophages were stimulated with ATP or Nigericin. The cells were immunostained with anti-NLRP3 and anti-Cullin1 antibodies. The supernatants were analyzed by ELISA for IL-1β secretion. Data shown are means ± SEM, *p<0.05, **p<0.01, ***p<0.0001. Data information: In ( A–G ), the cell lysates were immunoprecipitated with anti-HA antiboody (A), anti-Flag antibody (B, C), anti-NLRP3 antibody (D, F), or anti-Cullin1 antibody (E, G) and then immunoblotted with indicated antibodies.

Article Snippet: Human IL-1β/IL-1F2 Quantikine ELISA Kit (Cat# DLB50) was purchased from R&D Systems (Minnesota, USA).

Techniques: Transfection, Infection, Marker, Confocal Microscopy, Enzyme-linked Immunosorbent Assay, Immunoprecipitation

Journal: bioRxiv

Article Title: Cullin1 represses systematic inflammasome activation by binding and catalyzing NLRP3 ubiquitination

doi: 10.1101/289637

Figure Lengend Snippet: A HEK293T cells were transfected with pcDNA3.1(+)-NLRP3 and pCAGGS-HA-Ub at different concentrations. B HEK293T cells were transfected with pcDNA3.1(+)-NLRP3, pcDNA3.1(+)-HA-Ub, and/or pcDNA3.1(+)-3Flag-Cullin1, respectively. C HEK293T cells were transfected with pcDNA3.1(+)-NLRP3, pcDNA3.1(+)-HA-Ub, pcDNA3.1(+)-3Flag-Cullin1 or pcDNA3.1(+)-3Flag-ROC1, as indicated. D Daigrams of full-length NLRP3 and its point mutants. The potential ubiquitination sites in NLRP3 among the Lys residues were predicated by using UbPred software. One Lys (Lys689) is high confidence and six Lys (Lys93, Lys192, Lys194, Lys324, Lys430, and Lys696) are medium confidence. Seven mutations of CUL1 (K93R, K192R, K194R, K324R, K430R, K689R, and K696R), in which the K residues were replaced by R residues. E HEK293T cells were co-transfected with pcDNA3.1(+)-HA-Ub and pcDNA3.1(+)-Cullin1, along with pcDNA3.1(+)-3xFlag-NLRP3 or its mutants, respectively. F HEK293T cells were co-transfected with pcDNA3.1(+)-NLRP3 and/or pcDNA3.1(+)-HA-Ub, pcDNA3.1(+)-3Flag-Cullin1, pcDNA3.1(+)-HA-K48 or pcDNA3.1(+)-HA-K63, as indicated. G PMA-differentiated THP-1 macrophages stably expressing sh-NC and sh-Cullin1#2 were infected with IAV H3N2. H HEK293T cells were transfected with pcDNA3.1(+)-3XFlag-NLRP3, pcDNA3.1(+)-3XFlag-ASC, pcDNA3.1(+)-3XFlag-pro-caspase-1, pcDNA3.1(+)-3XFlag-pro-IL-1β, or pcDNA3.1(+)-3xFlag-Cullin1, as indicated. I HEK293T cells were transfected with pCAGGS-HA-NLRP3 and/or pcDNA3.1(+)-3XFlag-Cullin1, or pcDNA3.1(+)-3XFlag-ROC1. Data information: In ( A–C ), the lysates were immunoprecipitated with anti-NLRP3 antibody and immunoblotted with indicated antibodies. In ( E–I ), the lysates were immunoprecipitated with anti-Flag antibody (E) or anti-NLRP3 antibody (F, G) and then immunoblotted with indicated antibodies. The lysates were immunoblotted with the indicated antibodies (H, I).

Article Snippet: Human IL-1β/IL-1F2 Quantikine ELISA Kit (Cat# DLB50) was purchased from R&D Systems (Minnesota, USA).

Techniques: Transfection, Software, Stable Transfection, Expressing, Infection, Immunoprecipitation

Journal: bioRxiv

Article Title: Cullin1 represses systematic inflammasome activation by binding and catalyzing NLRP3 ubiquitination

doi: 10.1101/289637

Figure Lengend Snippet: A L929 cells were transfected with siR-NC (25 nM), siR-Cullin1#1 (25 nM), siR-Cullin1#2 (25 nM), or siR-Cullin1#3 (25 nM). Cullin1 and GAPDH mRNAs were determined by qRT-PCR (left) and CUL1 and GAPDH proteins were detected Western blot analyses (right). B C57BL/6 mice were injected with siR-NC (5 nM), siR-Cullin1#1 (5 nM), or siR-Cullin1#1 (10 nM) for 60 h, and the peritonitis of treated mice were then injected with Alum for 12 h. The endogenous mCUL1, mNLRP3, and mGAPDH proteins in the mice peritoneal exudates cells (PECs) were detected by Western blot analyses (left). Secreted IL-1β in the mice sera were analyzed by ELISA (right). C C57BL/6 mice (six mice per group) were injected with siR-NC or siR-Cullin1#1 for 60 h, and the peritonitis of treated mice were then injected with Alum. Secreted IL-1β in mice peritoneal lavage fluid were analyzed by ELISA. D C57BL/6 mice (six mice per group) were injected with siR-NC or siR-Cullin1#1 for 60 h, and the peritonitis of treated mice were then injected with Alum. Secreted IL-6 in mice peritoneal lavage fluid was analyzed by ELISA. E C57BL/6 mice (six mice per group) were injected with siR-NC or siR-Cullin1#1 for 60 h, and the peritonitis of treated mice were then injected with Alum. The total numbers of PECs in peritoneal cavity were stimulated. F C57BL/6 mice (six mice per group) were injected with siR-NC or siR-Cullin1#1 for 60 h, and the peritonitis of treated mice were then injected with Alum. The total numbers of Neutrophils (F) in peritoneal cavity were stimulated. G C57BL/6 mice (six mice per group) were injected with siR-NC or siR-Cullin1#1 for 60 h, and the peritonitis of treated mice were then injected with Alum. Histopathological changes in the mice spleen tissues were examined by H&E staining. Data information: In ( A–F ), data shown are means ± SEM, *P<0.05, **P<0.01, ***P<0.0001.

Article Snippet: Human IL-1β/IL-1F2 Quantikine ELISA Kit (Cat# DLB50) was purchased from R&D Systems (Minnesota, USA).

Techniques: Transfection, Quantitative RT-PCR, Western Blot, Injection, Enzyme-linked Immunosorbent Assay, Staining

Journal: bioRxiv

Article Title: Cullin1 represses systematic inflammasome activation by binding and catalyzing NLRP3 ubiquitination

doi: 10.1101/289637

Figure Lengend Snippet: A In resting cells and under normal physiological conditions, CUL1 interacts with NLRP3 to disrupt the NLRP3 inflammasome assembly, and catalyzes NLRP3 ubiquitination to repress the NLRP3 inflammasome activation. B In response to and inflammasome stimuli and pathogen infection, CUL1 disassociates from NLRP3 to release the repression of NLRP3 inflammasome assembly and activation. ASC binds NLRP3 to form ASC oligomers, which provides a platform for activation of pro-Casp-1 that regulates the maturation and secretion of IL-1β and IL-18, which in turn initiate multiple signaling pathways and drive inflammatory responses.

Article Snippet: Human IL-1β/IL-1F2 Quantikine ELISA Kit (Cat# DLB50) was purchased from R&D Systems (Minnesota, USA).

Techniques: Activation Assay, Infection

Journal: Developmental cell

Article Title: In vitro and in vivo development of the human airway at single cell resolution

doi: 10.1016/j.devcel.2020.01.033

Figure Lengend Snippet: A) Schematic of lung epithelial development. As the airways extend, bud tip progenitors are maintained as progenitors in the tips of branching buds, and leave cells behind that give rise to the intrapulmonary airways. Late in development, remaining bud tip progenitors differentiate into alveolar cells. No bud tip progenitors are present in the adult lung. B) Schematic of creation of epithelium-only bud tip progenitor organoids from 12 week fetal lungs. C) Feature plots for bud tip progenitor marker genes SFTPC, ID2 and HMGA1 from scRNA-seq of day 0 bud tip progenitor organoids maintained for 3 weeks in culture. Additional feature plots from this same data set are shown in Figure 4A. D) mRNA expression by QRT-PCR of basal cell marker TP63 in bud tip progenitor organoids treated for 3 days with serum-free basal medium supplemented with DMSO (control) or with signaling factors known to be important for lung development and cellular differentiation. DMSO (1:1,000 dilution), FGF7 (10 ng/mL), ‘3F’ (FGF7 10ng/mL, CHIR99021 3μM, ATRA 50 nM), Dexamethasone (25 ng/mL), IL6 (10 ng/mL), IL2 (50 U/mL), Smoothened Agonist (SAG; 500 nM), EGF (100 ng/mL), FGF10 (500 ng/mL), CHIR99021 (2 μM), IFNγ (10 ng/mL), TGFβ1 (100 ng/mL), BMP4 (100 ng/mL), DAPT (10 μM), Hydrocortisone (100 ng/mL). Gene expression is reported as arbitrary units. Treatment with TGFβ1 led to a significant increase in the expression of TP63 (one-way Analysis of Variance (ANOVA) (alpha=0.05, p<0.0001, F=14.7. Dunnett’s test of multiple comparison’s compared the mean of each group to the mean of the DMSO control group.) Estimated p values are shown on the graph. Error bars are plotted to show mean +/− the standard error of the mean. N=3 independent biological specimens. Data is from a single experiment. E) Bud tip progenitor organoids were treated with FGF7 (10 ng/mL), a permissive environment for TP63 expression compared to maintenance in bud tip progenitor medium (‘3F’), or with FGF7 (10 ng/mL) plus factors to inhibit SMAD signaling (A8301 [1 μM] and NOGGIN [100 ng/mL]) and TP63 gene expression was evaluated by QRT-PCR after 10 days in culture. A one-way Analysis of Variance was used followed by Tukey’s multiple comparison test to compare the means of each group to the mean of every other group. Estimated p values are reported on the graph. Error bars are plotted to show mean +/− the standard error of the mean. N=3 independent biological specimens. Data is from a single experiment. F) Bud tip progenitor organoids were treated for 3 days with SMAD activation or inhibition conditions and expression of TP63 was evaluated by QRT-PCR for all treatment groups. All media contained 3F components (FGF7 10ng/mL, CHIR99021 3μM, ATRA 50 nM), with individual groups containing combinations of: DMSO (1:1000 dilution), TGFβ1 (100 ng/mL), BMP4 (100 ng/mL), SB431542 (10 μM), LDN212854 (200 nM). One-way ANOVA alpha=0.05, F=21.19, p<0.0001; Tukey’s multiple comparisons of the mean of each group versus the mean in all other groups, estimated p values are reported on the graph. 3 days TGFβ1 and BMP4 is referred to as ‘dual SMAD activation’, or ‘DSA’. Data is plotted as arbitrary units. Error bars are plotted to show mean +/− the standard error of the mean. N=3 independent biological specimens. Data is from a single experiment and is representative of n=3 experiments. G) QRT-PCR for markers of canonical differentiated lung epithelial cell types showing DMSO (gray bars) and DSA treated (blue bars) organoids after 3 total days of treatment. Data is plotted as fold change over DMSO controls. Two-sided Mann-Whitney Tests were performed to compare the mean of the DMSO group to the Dual Smad Activation group (alpha=0.05). Error bars represent the mean +/− the standard error of the mean. n=3 independent biological specimens, and data is from a single experiment and is representative of n=3 experiments. H) Protein staining of DMSO treated (control) fetal bud tip progenitor organoids (top row) and 3 days of DSA treatment (bottom row) for TP63+ protein (green), KRT5 (pink) and DAPI (blue). Scale bar represents 50 μm. I) Quantification of (g). Total number of TP63+ cells were counted for 3–9 individual organoids across 3 biological replicates. n=3 independent biological specimens. A two-sided Mann Whitney test (alpha=0.05) was used to compare the means of each sample. For all graphs, p values are reported as follows: * p<0.05; ** p<0.01, *** p<0.001, **** p<0.0001.

Article Snippet: Growth factors and small molecules were used at the following concentrations: FGF10 (500 ng/mL, made in-house as previously described), A8301 (1 μM, Stem Cell Technologies cat. no. 72024), NOGGIN (100 ng/mL, R&D Systems, cat. no. 6057), Y27632 (APExBIO cat. no. A30008), LDN212854 (200 nM, R&D Systems cat. no. 6151/10), SB431542 (10 μM, Stemgent cat. no. 04–0010), TGFβ1(100 ng/mL, R&D systems cat. no. 240-B-002), BMP4 (100 ng/mL, R&D systems cat. no. 314-BP-050) Dexamethasone (25 ng/mL, Stem Cell Technologies cat. no. 72092), IL6 (10 ng/mL, R&D Systems, cat. no. 206-IL-010), IL2 (50 U/mL, R&D Systems, cat. no. 202-IL-010), Smoothened Agonist (SAG; 500 nM, R&D Systems, cat. no. 4366/1), EGF (100 ng/mL, R&D Systems cat. no. 236-EG-200), IFNγ (10 ng/mL, R&D Systems cat. no. {"type":"entrez-protein","attrs":{"text":"CAA31639","term_id":"32692","term_text":"CAA31639"}} CAA31639 ), DAPT (10 μM, R&D Systems cat. no. 2634/10), Hydrocortisone (100 ng/mL, Stem Cell Technologies cat. no. 74142).

Techniques: Marker, Expressing, Quantitative RT-PCR, Control, Cell Differentiation, Gene Expression, Comparison, Activation Assay, Inhibition, MANN-WHITNEY, Staining

Journal: Developmental cell

Article Title: In vitro and in vivo development of the human airway at single cell resolution

doi: 10.1016/j.devcel.2020.01.033

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Growth factors and small molecules were used at the following concentrations: FGF10 (500 ng/mL, made in-house as previously described), A8301 (1 μM, Stem Cell Technologies cat. no. 72024), NOGGIN (100 ng/mL, R&D Systems, cat. no. 6057), Y27632 (APExBIO cat. no. A30008), LDN212854 (200 nM, R&D Systems cat. no. 6151/10), SB431542 (10 μM, Stemgent cat. no. 04–0010), TGFβ1(100 ng/mL, R&D systems cat. no. 240-B-002), BMP4 (100 ng/mL, R&D systems cat. no. 314-BP-050) Dexamethasone (25 ng/mL, Stem Cell Technologies cat. no. 72092), IL6 (10 ng/mL, R&D Systems, cat. no. 206-IL-010), IL2 (50 U/mL, R&D Systems, cat. no. 202-IL-010), Smoothened Agonist (SAG; 500 nM, R&D Systems, cat. no. 4366/1), EGF (100 ng/mL, R&D Systems cat. no. 236-EG-200), IFNγ (10 ng/mL, R&D Systems cat. no. {"type":"entrez-protein","attrs":{"text":"CAA31639","term_id":"32692","term_text":"CAA31639"}} CAA31639 ), DAPT (10 μM, R&D Systems cat. no. 2634/10), Hydrocortisone (100 ng/mL, Stem Cell Technologies cat. no. 74142).

Techniques: Transduction, Control, Virus, Plasmid Preparation, Recombinant, RNAscope, Multiplex Assay, In Situ Hybridization, Generated, Software

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Effect of bone marrow mesenchymal stem cells-derived exosomes on diabetes-induced retinal injury: Implication of Wnt/ b-catenin signaling pathway.

doi: 10.1016/j.biopha.2022.113554

Figure Lengend Snippet: Fig. 3. Effect of BM MSC-derived exosomes intravitreal administration on wnt/b-catenin pathway in diabetic rats. MSC-derived exosomes were intravitreally injected to eight weeks’ diabetic rats in a single dose of 0.5 ml for each rat at a concentration (100 µg protein /ml) alone or in combination with wnt3a (20 ng). Additional group received DKK1 3 μl. Western blot was used to evaluate average relative density of phosphorylated b-catenin and total b- catenin (A) LDL Receptor Related Protein 6 (LRP6) and Phospho-LRP6 (B) in retinal tissues of various experimental groups. * Represents significance in comparison with control group at p˂0.05, * * represents significance in comparison with control group at p˂0.01, $ represents significance in comparison with diabetic group at p˂0.05, $$ represents significance in comparison with diabetic group at p˂0.01, ## represents significance in comparison with wnt3a group at p˂0.01. Data are expressed as mean±SE, (n = 6).

Article Snippet: Recombinant Mouse Wnt3a (Catalog Number: 1324-WN, R&D Systems, Bio-techno brand); Wnt3a 40 μg/ml.

Techniques: Derivative Assay, Injection, Concentration Assay, Western Blot, Comparison, Control

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Effect of bone marrow mesenchymal stem cells-derived exosomes on diabetes-induced retinal injury: Implication of Wnt/ b-catenin signaling pathway.

doi: 10.1016/j.biopha.2022.113554

Figure Lengend Snippet: Fig. 4. Effect of BM MSC-derived exosomes intravitreal administration on wnt/b-catenin induced oxidative stress in diabetic rats. MSC-derived exosomes were intravitreally injected to eight weeks diabetic rats in a single dose of 0.5 ml for each rat at a concentration (100 µg protein /ml) alone or in combination with wnt3a (20 ng). A further group of diabetic rats received DKK1. RT-PCR was used to evaluate mRNA levels of NADPH oxidase 2 (Nox2) and Nox4 (A), superoxide dismutase 1 (SOD1), (SOD2) in retinal tissues of various experimental groups (B). Data are expressed as median (maximum and minimum). SOD activity was assayed in retinal tissue by colorimetric assay (C), and NOX1 concentration was assayed by ELISA method (D). Data are expressed as mean±SE. * represents significance in comparison with control group at p˂0.05, ** represents significance in comparison with control group at p˂0.01, $ represents significance in comparison with diabetic group at p˂0.05, $$ represents significance in comparison with diabetic group at p˂0.01, ## represents significance in comparison with wnt3a group at p˂0.01, (n = 6).

Article Snippet: Recombinant Mouse Wnt3a (Catalog Number: 1324-WN, R&D Systems, Bio-techno brand); Wnt3a 40 μg/ml.

Techniques: Derivative Assay, Injection, Concentration Assay, Reverse Transcription Polymerase Chain Reaction, Activity Assay, Colorimetric Assay, Enzyme-linked Immunosorbent Assay, Comparison, Control

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Effect of bone marrow mesenchymal stem cells-derived exosomes on diabetes-induced retinal injury: Implication of Wnt/ b-catenin signaling pathway.

doi: 10.1016/j.biopha.2022.113554

Figure Lengend Snippet: Fig. 5. Effect of BM MSC-derived exosomes intravitreal administration on wnt/b-catenin triggered inflammation in diabetic rats. MSC-derived exosomes were intravitreally injected to eight weeks’ diabetic rats in a single dose of 0.5 ml for each rat at a concentration (100 µg protein /ml) alone or in combination with wnt3a (20 ng). A further group of diabetic rats received DKK1. RT-PCR was used to evaluate mRNA levels of c-Myc and CyclinD1 (A), tumor necrosis factor-α (TNF-α) and intercellular adhesion molecule-1 (ICAM-1) (B), vascular endothelial growth factor (VEGF) and vascular endothelial-cadherin (VE-cadherin) (C) in retinal tissues of various experimental groups. * represents significance in comparison with control group at p˂0.05, * * represents significance in comparison with control group at p˂0.01, $ represents significance in comparison with diabetic group at p˂0.05, $$ represents significance in comparison with diabetic group at p˂0.01, ## represents significance in comparison with wnt3a group at p˂0.01. Data are expressed as median (maximum and minimum), (n = 6).

Article Snippet: Recombinant Mouse Wnt3a (Catalog Number: 1324-WN, R&D Systems, Bio-techno brand); Wnt3a 40 μg/ml.

Techniques: Derivative Assay, Injection, Concentration Assay, Reverse Transcription Polymerase Chain Reaction, Comparison, Control

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Effect of bone marrow mesenchymal stem cells-derived exosomes on diabetes-induced retinal injury: Implication of Wnt/ b-catenin signaling pathway.

doi: 10.1016/j.biopha.2022.113554

Figure Lengend Snippet: Fig. 6. Effect of BM MSC-derived exosomes intravitreal administration on miR- 129–5 P& miR-34a expression levels. Quantitative analysis for relative expression of miR-129–5 P& miR-34a. * represents significance in comparison with control group at p˂0.05, ** represents significance in comparison with control group at p˂0.01, $ represents significance in comparison with diabetic group at p˂0.05, $$ represents significance in comparison with diabetic group at p˂0.01, ## represents significance in comparison with wnt3a group at p˂0.01. Data are expressed as mean±SE, (n = 6).

Article Snippet: Recombinant Mouse Wnt3a (Catalog Number: 1324-WN, R&D Systems, Bio-techno brand); Wnt3a 40 μg/ml.

Techniques: Derivative Assay, Expressing, Comparison, Control

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Effect of bone marrow mesenchymal stem cells-derived exosomes on diabetes-induced retinal injury: Implication of Wnt/ b-catenin signaling pathway.

doi: 10.1016/j.biopha.2022.113554

Figure Lengend Snippet: Fig. 8. Representative photomicrographs of iNOS immuno-stained sections from retinal tissues. (A) Control groups (group I) showed no immunoreactivity for iNOS. (B, C, and F): Groups IIa, IIb and Wnt3a revealed strong positive cytoplasmic iNOS immunoreactivity in GCL and INL. (D and E): Groups III and IV minimal cytoplasmic iNOS immunoreactivity within the GCL and INL. (G): Group VI revealed mild positive cytoplasmic iNOS immunoreactivity of GCL and INL. (H): His togram representing the mean area percentage of iNOS immuno-reaction in all experimental groups. * Represents significance in comparison with control group at p˂0.05, ** represents significance in comparison with control group at p˂0.01, $ represents significance in comparison with diabetic group at p˂0.05, $$ represents

Article Snippet: Recombinant Mouse Wnt3a (Catalog Number: 1324-WN, R&D Systems, Bio-techno brand); Wnt3a 40 μg/ml.

Techniques: Staining, Control, Comparison

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Effect of bone marrow mesenchymal stem cells-derived exosomes on diabetes-induced retinal injury: Implication of Wnt/ b-catenin signaling pathway.

doi: 10.1016/j.biopha.2022.113554

Figure Lengend Snippet: Fig. 9. VEGF immuno-stained retinal tissues. (A) Control groups (group I) showed weak immunoreactivity for VEGF. (B, C, and F): Groups IIa, IIb, and V revealed strongly positive VEGF immunoreactivity in NFL, GCL, INL, and OPL. (D and E): Groups III and IV showed minimal VEGF immunoreactivity within the NFL, GCL, INL and OPL. (G): Group VI revealed mild positive VEGF immunoreactivity of NFL, GCL, INL and OPL. (H): Histogram representing the mean area percentage of VEGF immuno-reaction in all experimental groups. * represents significance in comparison with control group at p˂0.05, ** represents significance in comparison with control group at p˂0.01, $ represents significance in comparison with diabetic group at p˂0.05, $$ represents significance in comparison with diabetic group at p˂0.01, ## represents significance in comparison with wnt3a group at p˂0.01. Data are expressed as mean±SE, (n = 6).

Article Snippet: Recombinant Mouse Wnt3a (Catalog Number: 1324-WN, R&D Systems, Bio-techno brand); Wnt3a 40 μg/ml.

Techniques: Staining, Control, Comparison

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Effect of bone marrow mesenchymal stem cells-derived exosomes on diabetes-induced retinal injury: Implication of Wnt/ b-catenin signaling pathway.

doi: 10.1016/j.biopha.2022.113554

Figure Lengend Snippet: Fig. 10. GFAP immuno-stained sections from retinal tissues. (A) Control groups (group I) showed weak immunoreactivity for GFAP very close to GCL. (B, C, and F): Groups IIa, IIb, and V revealed strongly positive GFAP immunoreactivity in NFL and GCL, which was more prominent around blood capillaries in the innermost layers of retina and in Muller cell processes. (D and E): Groups III and IV showed minimal GFAP immunoreactivity within the Muller cell end feet. (G): Group VI revealed mildly positive GFAP immunoreactivity of Muller cell bodies and their processes. (H): Histogram representing the mean area percentage of GFAP immuno-reaction in all experimental groups. * represents significance in comparison with control group at p˂0.05, ** represents significance in comparison with control group at p˂0.01, $ represents significance in comparison with diabetic group at p˂0.05, $$ represents significance in comparison with diabetic group at p˂0.01, ## represents significance in comparison with wnt3a group at p˂0.01. Data are expressed as mean±SE, (n = 6).

Article Snippet: Recombinant Mouse Wnt3a (Catalog Number: 1324-WN, R&D Systems, Bio-techno brand); Wnt3a 40 μg/ml.

Techniques: Staining, Control, Comparison

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Effect of bone marrow mesenchymal stem cells-derived exosomes on diabetes-induced retinal injury: Implication of Wnt/ b-catenin signaling pathway.

doi: 10.1016/j.biopha.2022.113554

Figure Lengend Snippet: Fig. 11. NF-κB P65 immuno-stained sections from the retina. (A) Control groups (group I) showed no immunoreactivity for NF-κB P65. (B, C, and F): Groups IIa, IIb, and V revealed strongly positive NF-κB P65 immunoreactivity in GCL and INL. (D and E): Groups III and IV showed minimal NF-κB P65 immunoreactivity within GCL and INL. (G): Group VI revealed mildly positive NF-κB P65 immunoreactivity of GCL and INL. (H): Histogram representing immunoreactive score for NF-κB/p65 differentially in the nucleus and cytoplasm. Data are expressed as mean ± SE (n = 6). ***: p < 0.001 versus normal, $$: p < 0.01, $$$: p < 0.001 versus diabetic group, ###: p < 0.001 versus diabetic+DKK1 group, ΦΦΦ: p < 0.001 versus diabetic+ MSC-derived exosomes group and ΔΔΔ: p < 0.001 versus wnt3a group for nuclear expression, @@@: p < 0.001 versus normal, αα: p < 0.01, ααα: p < 0.001 versus diabetic group, βββ: p < 0.001 versus diabetic+DKK1 group, γγγ: p < 0.001 versus diabetic+ MSC-derived exosomes group and ΨΨ: p < 0.001 versus wnt3a group for cytoplasmic expression.

Article Snippet: Recombinant Mouse Wnt3a (Catalog Number: 1324-WN, R&D Systems, Bio-techno brand); Wnt3a 40 μg/ml.

Techniques: Staining, Control, Derivative Assay, Expressing

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Effect of bone marrow mesenchymal stem cells-derived exosomes on diabetes-induced retinal injury: Implication of Wnt/ b-catenin signaling pathway.

doi: 10.1016/j.biopha.2022.113554

Figure Lengend Snippet: Fig. 14. Figure a-b: Fundus photo of a normal (Group I) retinal rat shows: normal retinal appearance, optic nerve head and vasculature. OCT scan of the retinal layers shows: normal appearance and thickness of all the retinal layers. Figure c-d: Fundus photo of a diabetic (Group II) retinal rat shows obscuration of the retinal appearance due to vitreous hemorrhage and veinous beading (irregular dilatation of veins). OCT scan of the retinal layers shows: irregular appearance and reduction of all the retinal layers thickness due to neurological degeneration. Figure e-f: Fundus photo of retina from DKK1 treated rat (Group III) shows within normal retinal appearance, optic nerve head and vasculature. OCT scan of the retinal layers shows: within normal appearance and reduction of the inner retinal layers thickness. Figure g-h: Fundus photo of retinal rat from exosomes treated group (group IV) shows normal retinal appearance, optic nerve head and vasculature. OCT scan of the retinal layers shows: within normal appearance and thickness of all the retinal layers. Figure i-j: Fundus photo of Group V (Wnt3a group) retinal rat shows obscuration of the retinal appearance due to vitreous hemorrhage and attenuation of the retinal vasculature. OCT scan of the retinal layers shows: irregular appearance and reduction of all the retinal layers thickness due to neurological degeneration. Figure k-L: Fundus photo of Wnt3a and exosomes (group VI) retinal rat shows obscuration of the retinal appearance due to vitreous hemorrhage and attenuation of the retinal vasculature. OCT scan of the retinal layers shows: irregular appearance and reduction of all the retinal layers thickness due to neurological degeneration. Figure M: retinal thickness (micron).

Article Snippet: Recombinant Mouse Wnt3a (Catalog Number: 1324-WN, R&D Systems, Bio-techno brand); Wnt3a 40 μg/ml.

Techniques: