Journal: Cell

Article Title: The 7q11.23 Protein DNAJC30 is an Auxiliary Component of ATP Synthase and Links Mitochondria to Brain Development

doi: 10.1016/j.cell.2018.09.014

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Avidin-Biotin blocker , Vector Laboratories , Cat#SP-2001.

Techniques: Recombinant, Protease Inhibitor, Modification, Avidin-Biotin Assay, Plasmid Preparation, Bicinchoninic Acid Protein Assay, Knock-Out, Sequencing, Clone Assay, Software, Microscopy, Digital PCR, Fluorescence, Transmission Assay

Journal: Cell Proliferation

Article Title: Haematopoietic lineage‐committed bone marrow cells, but not cloned cultured mesenchymal stem cells, contribute to regeneration of renal tubular epithelium after HgCl 2 ‐induced acute tubular injury

doi: 10.1111/j.1365-2184.2008.00545.x

Figure Lengend Snippet: Examples of engraftment of haematopoietic lineage marrow cells (HLMCs) and mesenchymal stem cells at 3 days after HgCl2. (a) GFP‐positive control shows proximal tubular cells (PHA‐L, blue colour) co‐stained for GFP (brown colour). (b) GFP‐negative control (wild type) showing no detection of GFP. (c) BMT mouse treated with HgCl2 demonstrating GFP‐positive proximal tubular epithelial cells. Dashed black arrows indicate PHA‐L stained donor HLMC‐derived tubular cells. (d) Male positive control; Y chromosomes were detected by indirect FISH, black arrows point to Y chromosomes (brown dot) in proximal tubular cells histochemically stained with PHA‐L (red colour). (e) Female control showing the lack of Y chromosome detection within proximal tubular cells histochemically stained with PHA‐L. (f) Scattered Y chromosomes (brown dot) in renal interstitial area, not in proximal tubular cells. (g) High magnification of yellow box in (f); a–f, ×400; g, ×600. BMT, bone marrow transplantation; FISH, fluorescence in situ hybridization; PHA‐L, Phaseolus vulgaris leucoagglutinin.

Article Snippet: After GFP staining, tissue sections were microwaved in 10 m m tri‐sodium citrate (pH 6.0) for 10 min and biotin was blocked using a biotin blocking kit (DAKO), and then incubated for 45 min with biotinylated Phaseolus vulgaris leucoagglutinin (PHA‐L) 1/100 (B‐1115, Vector Laboratories, Burlingame, CA, USA) to detect proximal convoluted tubules, or biotinylated peanut agglutinin (PNA) 1/800 (B‐1075, Vector Laboratories) to detect distal convoluted tubules.

Techniques: Positive Control, Staining, Negative Control, Derivative Assay, Transplantation Assay, Fluorescence, In Situ Hybridization

Journal: Cell Proliferation

Article Title: Haematopoietic lineage‐committed bone marrow cells, but not cloned cultured mesenchymal stem cells, contribute to regeneration of renal tubular epithelium after HgCl 2 ‐induced acute tubular injury

doi: 10.1111/j.1365-2184.2008.00545.x

Figure Lengend Snippet: (a) Changes in the abundance of GFP‐positive cells within the PHA‐L stained cell population in control mice and mice treated with HgCl2 (n = 5 per treatment time point). *P < 0.05 versus the same group at day 0; +P < 0.05 versus group CON at the corresponding time point. The percentages of GFP‐positive cells were adjusted based on a correction factor derived from the actual cell count of 41% of PHA‐L stained cells being GFP‐positive in GFP donor mice. (b) Changes in the 3H‐LI of PHA‐L stained proximal tubular cells of: combined indigenous and donor HLMC (left panel); indigenous origin (central panel); donor HLMC origin (right panel). n = 5 per group. *P < 0.05 versus the same group at day 0; +P < 0.05 versus group CON at the corresponding time point. (c–g) Examples of chimerism and proliferation (3H‐thymidine labelling) of GFP+ HLMC‐derived PHA‐L‐stained cells at 3 days after HgCl2. (c) Black arrowheads indicate PHA‐L stained (blue colour in tubular cell apical membrane) donor GFP+ HLMC‐derived tubular cells (brown colour in cytoplasm) under bright field, and black arrows indicate 3H‐thymidine labelling of PHA‐L‐stained HLMC‐derived tubular cells, ×500. (d) The same field under dark field, white arrowheads point to silver grains (3H‐thymidine labelling) and white arrows indicate 3H‐thymidine labelling of PHA‐L‐stained HLMC‐derived tubular cells (×500). (e) Bright field and (f) dark field are the higher magnification of yellow box area in (c). (g) The images in (e) and (f) were combined to help to show silver grains over cells that are donor GFP+ HLMC‐derived PHA‐L‐stained tubular cells. GFP, green fluorescent protein; HLMC, haematopoietic lineage marrow cell; PHA‐L, Phaseolus vulgaris leucoagglutinin.

Article Snippet: After GFP staining, tissue sections were microwaved in 10 m m tri‐sodium citrate (pH 6.0) for 10 min and biotin was blocked using a biotin blocking kit (DAKO), and then incubated for 45 min with biotinylated Phaseolus vulgaris leucoagglutinin (PHA‐L) 1/100 (B‐1115, Vector Laboratories, Burlingame, CA, USA) to detect proximal convoluted tubules, or biotinylated peanut agglutinin (PNA) 1/800 (B‐1075, Vector Laboratories) to detect distal convoluted tubules.

Techniques: Staining, Derivative Assay, Cell Counting

Journal: Genes & Development

Article Title: Apoptosis of leukocytes triggered by acute DNA damage promotes lymphoma formation

doi: 10.1101/gad.1940210

Figure Lengend Snippet: Loss of Puma prevents compensatory proliferation of stem cells upon DNA damage. (A) To compare the rates of proliferation in different stem cell populations, the mice of the indicated genotpyes were exposed to IR, and, on days 3 and 7, were injected i.p. with a single dose of BrdU. Four hours later, mice were sacrificed, and the percentage of BrdU+ cells was assessed by flow cytometry in the individual stem cell subsets or total bone marrow. The relative increase in proliferation (BrdU+ cells) in relation to untreated controls was calculated by using the following equation: (IR-induced proliferation percent − spontaneous proliferation percent)/(100 − spontaneous proliferation percent). Bars represent fold induction of BrdU+ cycling cells as mean ± SEM of three to four animals per genotype and three independent experiments. (*) Significant differences in BrdU uptake were observed between wild-type and puma−/− or p53−/− MPP (P < 0.03) and LT-HSC (P < 0.01) subsets at days 3 or 7 post-IR. (B) Mice were fed BrdU in drinking water for 12 d, yielding between 70% and 90% of labeling efficiency in both genotypes alike. Then mice were exposed to 1.75 Gy IR at days 0 and 7. The percentage of BrdU+ stem cell subsets was assessed by flow cytometry. IR-induced loss of BrdU as an indirect measure of proliferation was calculated by subtracting the percentage of BrdU+ cells of mice pre-exposed to IR from the percentage of BrdU+ cells in control animals. Symbols represent mean ± SEM of three to four animals per genotype and time point. Spontaneous BrdU loss was not different between genotypes (P > 0.78). (*) BrdU loss was significantly different at all time points after IR in MPP (P < 0.014) and LSK (P < 0.026) cells at days 3 and 7, and in LT-HSCs (P < 0.014) at days 7 and 10. (C) Cell cycle analysis using Ki67 was performed in control mice or mice exposed to IR 7 d before. The percentage of cells in G0 is plotted. Bars represent mean ± SEM of three animals per genotype. (*) The percentage of stem/progenitor cells in G0 was significantly higher in puma+/−, puma−/−, and p53−/− when compared with wild-type mice (P < 0.04).

Article Snippet: HSC populations were defined using a biotin-labeled lineage marker flow cytometry kit (e-Bioscience).

Techniques: Injection, Flow Cytometry, Labeling, Cell Cycle Assay

Journal: International Journal of Molecular Sciences

Article Title: Mulberry Component Kuwanon C Exerts Potent Therapeutic Efficacy In Vitro against COVID-19 by Blocking the SARS-CoV-2 Spike S1 RBD:ACE2 Receptor Interaction

doi: 10.3390/ijms232012516

Figure Lengend Snippet: The inhibition of the interaction between the spike protein S1 receptor-binding domain (RBD) and the human angiotensin-converting enzyme 2 (ACE2) receptor by kuwanon C (KC). Spike protein coated on a 96-well plate interacted with a preincubated mixture of the ACE2 receptor and ( A ) anti-SARS-CoV-2 spike S1 antibody as the positive control and ( B ) 0, 3.125, 6.25, 12.5, 25, 50, or 100 μM KC. The inhibition of the spike S1 RBD:ACE2 receptor interaction by KC was determined based on chemiluminescence measurements.

Article Snippet: The SARS-CoV-2 spike/ACE2 inhibitor screening assay kit, biotin-labeled recombinant protein ACE2 receptor, and spike protein S1 RBD (BPS Bioscience, San Diego, CA, USA) were purchased for the competitive ELISA assay and BLItz analysis.

Techniques: Inhibition, Binding Assay, Positive Control

Journal: International Journal of Molecular Sciences

Article Title: Mulberry Component Kuwanon C Exerts Potent Therapeutic Efficacy In Vitro against COVID-19 by Blocking the SARS-CoV-2 Spike S1 RBD:ACE2 Receptor Interaction

doi: 10.3390/ijms232012516

Figure Lengend Snippet: The global kinetic analysis of KC binding to biotinylated ( A ) spike S1 RBD- and ( B ) ACE2 receptor-immobilized BLI sensors. The kinetics for the binding of KC to the spike S1 RBD or the ACE2 receptor were measured by the association of 0, 50, 200, and 400 μM of KC in PBS containing 1% DMSO with immobilized spike S1 or ACE2 receptor and the subsequent dissociation in PBS containing 1% DMSO.

Article Snippet: The SARS-CoV-2 spike/ACE2 inhibitor screening assay kit, biotin-labeled recombinant protein ACE2 receptor, and spike protein S1 RBD (BPS Bioscience, San Diego, CA, USA) were purchased for the competitive ELISA assay and BLItz analysis.

Techniques: Binding Assay

Journal: International Journal of Molecular Sciences

Article Title: Mulberry Component Kuwanon C Exerts Potent Therapeutic Efficacy In Vitro against COVID-19 by Blocking the SARS-CoV-2 Spike S1 RBD:ACE2 Receptor Interaction

doi: 10.3390/ijms232012516

Figure Lengend Snippet: The binding kinetics of KC to spike S1 RBD and ACE2 receptor.

Article Snippet: The SARS-CoV-2 spike/ACE2 inhibitor screening assay kit, biotin-labeled recombinant protein ACE2 receptor, and spike protein S1 RBD (BPS Bioscience, San Diego, CA, USA) were purchased for the competitive ELISA assay and BLItz analysis.

Techniques: Binding Assay

Journal: International Journal of Molecular Sciences

Article Title: Mulberry Component Kuwanon C Exerts Potent Therapeutic Efficacy In Vitro against COVID-19 by Blocking the SARS-CoV-2 Spike S1 RBD:ACE2 Receptor Interaction

doi: 10.3390/ijms232012516

Figure Lengend Snippet: In silico docking simulation between KC and the spike protein/ACE2 receptor. KC was docked onto the SARS-CoV-2 spike protein and ACE2 receptor (PDB code: 6M0J) using AutoDock Vina. The pharmacophore of KC with each target proteins was analyzed using BIOVIA Discovery Studio Visualizer.

Article Snippet: The SARS-CoV-2 spike/ACE2 inhibitor screening assay kit, biotin-labeled recombinant protein ACE2 receptor, and spike protein S1 RBD (BPS Bioscience, San Diego, CA, USA) were purchased for the competitive ELISA assay and BLItz analysis.

Techniques: In Silico

Journal: International Journal of Molecular Sciences

Article Title: Mulberry Component Kuwanon C Exerts Potent Therapeutic Efficacy In Vitro against COVID-19 by Blocking the SARS-CoV-2 Spike S1 RBD:ACE2 Receptor Interaction

doi: 10.3390/ijms232012516

Figure Lengend Snippet: KC inhibits SARS-CoV-2 lentiviral pseudovirus infection in HEK293T cells stably expressing human ACE2 and TMPRSS2. ( A ) The cytotoxic effect of KC in HEK293T cells stably expressing human ACE2 and TMPRSS2 was determined using the MTT assay. HEK293T cells were cultured in 96-well plates (5 × 10 4 cells/well) for 18 h. ( B ) The ACE2 expression level in HEK293T cells was monitored during KC treatment using real-time quantitative PCR analysis. Then, WT or mutant (D614G) SARS-CoV-2 spike pseudovirus (at a final concentration of 1 × 10 4 TU/mL to each well) were mixed with different concentrations of KC (2 and 20 μM) or anti-SARS-CoV-2 antibody, and the mixtures were incubated at 37 °C for 1 h. Then, these mixtures were added to HEK293T cells. ( C , D ) Green fluorescent protein (GFP) expression levels using flow cytometry were assessed at 72 h after viral infection, scale bar = 100 μm. ( E , F ) The inhibitory effect of SARS-CoV-2 spike pseudovirus infection was determined by measuring GFP expression using flow cytometry and measured under a fluorescence microscope. Bar graph (mean ± SEM) statistics were determined from three experimental data sets using one-way ANOVA with Tukey’s post hoc test, *** p < 0.001, compared with the CON (KC-untreated) samples. ### p < 0.001, compared with the cell-only sample.

Article Snippet: The SARS-CoV-2 spike/ACE2 inhibitor screening assay kit, biotin-labeled recombinant protein ACE2 receptor, and spike protein S1 RBD (BPS Bioscience, San Diego, CA, USA) were purchased for the competitive ELISA assay and BLItz analysis.

Techniques: Infection, Stable Transfection, Expressing, MTT Assay, Cell Culture, Real-time Polymerase Chain Reaction, Mutagenesis, Concentration Assay, Incubation, Flow Cytometry, Fluorescence, Microscopy

Journal: International Journal of Molecular Sciences

Article Title: Mulberry Component Kuwanon C Exerts Potent Therapeutic Efficacy In Vitro against COVID-19 by Blocking the SARS-CoV-2 Spike S1 RBD:ACE2 Receptor Interaction

doi: 10.3390/ijms232012516

Figure Lengend Snippet: KC suppresses the infection of a clinical isolate of SARS-CoV-2 alpha strain (βCoV/Korea/KCDC03/2020) in Vero cells. Vero cells were cultured on 384-well plates (1.2 × 10 4 cells/well) for 24 h. Then, Vero cells were infected with SARS-CoV-2 (MOI 0.0125) immediately after being treated with serially diluted KC and incubated at 37 °C for 24 h. The cells were then stained using anti-SARS-CoV-2 nucleocapsid (N) primary antibody, Alexa Fluor 488-conjugated goat antirabbit IgG secondary antibody, and Hoechst 33342.

Article Snippet: The SARS-CoV-2 spike/ACE2 inhibitor screening assay kit, biotin-labeled recombinant protein ACE2 receptor, and spike protein S1 RBD (BPS Bioscience, San Diego, CA, USA) were purchased for the competitive ELISA assay and BLItz analysis.

Techniques: Infection, Cell Culture, Incubation, Staining

Journal: International Journal of Molecular Sciences

Article Title: Mulberry Component Kuwanon C Exerts Potent Therapeutic Efficacy In Vitro against COVID-19 by Blocking the SARS-CoV-2 Spike S1 RBD:ACE2 Receptor Interaction

doi: 10.3390/ijms232012516

Figure Lengend Snippet: Schematic of the blockade of the SARS-CoV-2 spike S1 RBD:ACE2 receptor interaction by KC.

Article Snippet: The SARS-CoV-2 spike/ACE2 inhibitor screening assay kit, biotin-labeled recombinant protein ACE2 receptor, and spike protein S1 RBD (BPS Bioscience, San Diego, CA, USA) were purchased for the competitive ELISA assay and BLItz analysis.

Techniques: