Journal: Cell Stress & Chaperones

Article Title: Recombinant production of ESAT-6 antigen in thermoinducible Escherichia coli : the role of culture scale and temperature on metabolic response, expression of chaperones, and architecture of inclusion bodies

doi: 10.1007/s12192-019-01006-x

Figure Lengend Snippet: Expression of rESAT-6 in E. coli ATCC® 53606™ by SDS-PAGE (18%) with a Coomassie staining (a,b) and Western blot (c,d) with anti-ESAT-6 antibody. Soluble (S), insoluble (I) protein fractions, and purified inclusion bodies (IBs) obtained at the end of the thermo-induced cultures (27 h) in shake flasks (a,c) and bioreactors (b,d) are presented. Lane 1, molecular weight standards (MW). Lanes 2 and 3, S and I fractions from non-induced culture at 30 °C, respectively. Lanes 4 and 5, S and IBs from thermo-induced culture at 39 °C, respectively. Lanes 6 and 7, S and IBs from thermo-induced culture at 42 °C, respectively. Arrowheads indicate the position of rESAT-6 between 10 and 15 kDa. The SDS-PAGE and Western blots were made for each culture independently, and the duplicates are shown in the supplementary material (Figs. ​(Figs.1S1S and ​and2S2S)

Article Snippet: In this work, we evaluate the effect of the culture scale (shake flasks and bioreactors) and induction temperature (39 and 42 °C) on the kinetic behavior of thermoinducible recombinant E. coli ATCC 53606 producing rESAT-6 (6-kDa early-secretory antigenic target from Mycobacterium tuberculosis ), compared with cultures grown at 30 °C (without induction).

Techniques: Expressing, SDS Page, Staining, Western Blot, Purification, Molecular Weight

Journal: Cell Stress & Chaperones

Article Title: Recombinant production of ESAT-6 antigen in thermoinducible Escherichia coli : the role of culture scale and temperature on metabolic response, expression of chaperones, and architecture of inclusion bodies

doi: 10.1007/s12192-019-01006-x

Figure Lengend Snippet: Immunoblotting of chaperones DnaK (a,b) and GroEL (c,d) in soluble (S), insoluble (I) protein fractions, and purified inclusion bodies (IBs) obtained in E. coli ATCC® 53606™ (rESAT-6) cultures. In shake flasks (a,c), lane 1, empty; lane 2, cell extract of wild-type E. coli 53606 grown at 42 °C as a control; lane 3, purified IBs harvested from shake flask culture induced at 39 °C with complex medium (LB) as a thermoinduction control; lane 4, molecular weight standards (MW); lanes 5 and 6, S and IBs from induced culture at 39 °C, respectively; lanes 7 and 8, S and I fractions from non-induced culture at 30 °C, respectively; and lanes 9 and 10, S and IBs from induced culture at 42 °C, respectively. In bioreactors, (b,d) lane 1, molecular weight standards (MW); lane 2, cell extract of wild-type E. coli 53606 grown at 42 °C as a control; lane 3, purified IBs harvested from culture induced at 39 °C with complex medium (LB); lanes 4 and 5, S and I fractions from non-induced culture at 30 °C, respectively; lanes 6 and 7, S and IBs from induced culture at 39 °C, respectively; and lanes 8 and 9, S and IBs from induced culture at 42 °C, respectively. Arrowheads indicate the position of chaperones DnaK (~ 70 kDa) and GroEL (~ 60 kDa)

Article Snippet: In this work, we evaluate the effect of the culture scale (shake flasks and bioreactors) and induction temperature (39 and 42 °C) on the kinetic behavior of thermoinducible recombinant E. coli ATCC 53606 producing rESAT-6 (6-kDa early-secretory antigenic target from Mycobacterium tuberculosis ), compared with cultures grown at 30 °C (without induction).

Techniques: Western Blot, Purification, Control, Molecular Weight

Journal: Cell Stress & Chaperones

Article Title: Recombinant production of ESAT-6 antigen in thermoinducible Escherichia coli : the role of culture scale and temperature on metabolic response, expression of chaperones, and architecture of inclusion bodies

doi: 10.1007/s12192-019-01006-x

Figure Lengend Snippet: Bacterial growth kinetics (a,b), glucose uptake (c,d) and acetate accumulation (e,f) of recombinant E. coli ATCC® 53606™ (rESAT-6) in 250 mL shake flasks (a,c,e) and 1.2 L bioreactors (b,d,f) at different induction temperatures. Non-induced culture as a control was kept at 30 °C (closed dots), and thermo-induced cultures were heated up to 39 °C (closed squares) and 42 °C (closed triangles). Thermoinduction (dotted line) of rESAT-6 production was started at OD600 nm of 1.4–2.0 AU (5 h) in shake flasks and 3.0–4.0 AU (6 h) in bioreactors. Average and standard deviation of at least two independent experiments are shown

Article Snippet: In this work, we evaluate the effect of the culture scale (shake flasks and bioreactors) and induction temperature (39 and 42 °C) on the kinetic behavior of thermoinducible recombinant E. coli ATCC 53606 producing rESAT-6 (6-kDa early-secretory antigenic target from Mycobacterium tuberculosis ), compared with cultures grown at 30 °C (without induction).

Techniques: Recombinant, Control, Standard Deviation

Journal: Cell Stress & Chaperones

Article Title: Recombinant production of ESAT-6 antigen in thermoinducible Escherichia coli : the role of culture scale and temperature on metabolic response, expression of chaperones, and architecture of inclusion bodies

doi: 10.1007/s12192-019-01006-x

Figure Lengend Snippet: Profiles of dissolved oxygen tension (a,b) and pH (c,d) of recombinant E. coli ATCC® 53606™ (rESAT-6). In shake flasks (a,c), non-induced cultures were kept at 30 °C (closed dots) and thermo-induced cultures were heated up to 39 °C (closed squares) and 42 °C (closed triangles). An average and standard deviation of at least two independent experiments are shown. In bioreactors (b,d), a typical trend is shown in solid line (─) of a non-induced culture kept at 30 °C, in dotted line (∙∙∙) of a thermo-induced culture at 39 °C, and in discontinuous line (---) of a thermo-induced culture at 42 °C. Thermoinduction of recombinant protein (vertical dotted line) was started at OD600 nm of 1.4–2.0 AU (5 h) in shake flasks and 3.0–4.0 AU (6 h) in bioreactors

Article Snippet: In this work, we evaluate the effect of the culture scale (shake flasks and bioreactors) and induction temperature (39 and 42 °C) on the kinetic behavior of thermoinducible recombinant E. coli ATCC 53606 producing rESAT-6 (6-kDa early-secretory antigenic target from Mycobacterium tuberculosis ), compared with cultures grown at 30 °C (without induction).

Techniques: Recombinant, Standard Deviation

Journal: Cell Stress & Chaperones

Article Title: Recombinant production of ESAT-6 antigen in thermoinducible Escherichia coli : the role of culture scale and temperature on metabolic response, expression of chaperones, and architecture of inclusion bodies

doi: 10.1007/s12192-019-01006-x

Figure Lengend Snippet: Growth and production parameters of recombinant E. coli ATCC®-53606 (rESAT-6) using two different culture scales (shake flasks and bioreactors) and two induction temperatures (39 and 42 °C). Non-induced culture (kept at 30 °C) was used as a control. Data show the mean and standard deviation for at least two independent experiments

Article Snippet: In this work, we evaluate the effect of the culture scale (shake flasks and bioreactors) and induction temperature (39 and 42 °C) on the kinetic behavior of thermoinducible recombinant E. coli ATCC 53606 producing rESAT-6 (6-kDa early-secretory antigenic target from Mycobacterium tuberculosis ), compared with cultures grown at 30 °C (without induction).

Techniques: Recombinant, Control, Standard Deviation

Journal: Gut Pathogens

Article Title: Development of multiplex PCR and multi-color fluorescent in situ hybridization ( m -FISH) coupled protocol for detection and imaging of multi-pathogens involved in inflammatory bowel disease

doi: 10.1186/s13099-018-0278-1

Figure Lengend Snippet: Specificity and sensitivity of the IS900 nPCR in DNA extracts from the modified DNAzol ® and the standard phenol/chloroform/isoamyl-alcohol DNA extraction protocols. nPCR based on the IS900 specific to MAP was performed on DNA template extracted by the standard phenol/chloroform/isoamyl-alcohol DNA extraction (I) and the modified DNAzol ® DNA extraction technique (II). A 298 bp fragment on 2% agarose gel is positive for MAP. a (1) Non-pathogenic E. coli strain K-12; (2) S. aureus ; (3) L. monocytogenes ; (4) K. pneumoniae ; (5) M. smegmatis ; (6) M. avium subspecies avium ; (7) M. xenopi ; (8) M. fortuitum subspecies fortuitum ; (9) MAP Clinical Strain JF7. b (1) MAP Strain 1; (2) MAP Strain 3; (3) MAP Strain 8B; (4) MAP Para 18; (5) MAP UCF3; (6) MAP UCF5; (7) MAP UCF7; (8) MAP Linda; (9) MAP MS137. c Serial dilution of MAP UCF4 DNA concentrations were analyzed by nPCR. (1) 31.7 ng/μL; (2) 3.17 ng/μL; (3) 317 pg/μL; (4) 31.7 pg/μL; (5) 3.17 pg/μL; (6) 317 fg/μL; (7) 31.7 fg/μL; (8) 3.17 fg/μL; (9) 317 ag/μL; (10) 31.7 ag/μL. +: MAP UCF4; N: No DNA; M: molecular weight marker

Article Snippet: Non - pathogenic Escherichia coli Strain K-12 , ATCC 8739.

Techniques: Modification, DNA Extraction, Agarose Gel Electrophoresis, Serial Dilution, Molecular Weight, Marker

Journal: Gut Pathogens

Article Title: Development of multiplex PCR and multi-color fluorescent in situ hybridization ( m -FISH) coupled protocol for detection and imaging of multi-pathogens involved in inflammatory bowel disease

doi: 10.1186/s13099-018-0278-1

Figure Lengend Snippet: Validation of multiplex PCR using individual and combined oligonucleotide primer sets and DNA extracts from mixed bacterial culture. Multiplex PCR was performed on DNA extracts from mixed bacterial cultures. DNA template was extracted using the modified DNAzol ® . (1) Non-pathogenic E. coli strain K-12 18s primers were used (171 bp); (2) MAP UCF4 IS900 AV1/AV2 primers were used (298 bp); (3) K. pneumoniae 23s primers were used (493 bp); (4) Mycobacterium avium complex (MAC) IS1311 primers were used (534 bp); (5) a cocktail of the 4 primer sets mentioned above were used. M: DNA molecular weight marker

Article Snippet: Non - pathogenic Escherichia coli Strain K-12 , ATCC 8739.

Techniques: Biomarker Discovery, Multiplex Assay, Modification, Molecular Weight, Marker

Journal: Gut Pathogens

Article Title: Development of multiplex PCR and multi-color fluorescent in situ hybridization ( m -FISH) coupled protocol for detection and imaging of multi-pathogens involved in inflammatory bowel disease

doi: 10.1186/s13099-018-0278-1

Figure Lengend Snippet: Multiplex PCR and m -FISH results for intestinal tissue from IBD

Article Snippet: Non - pathogenic Escherichia coli Strain K-12 , ATCC 8739.

Techniques: Multiplex Assay

Journal: Gut Pathogens

Article Title: Development of multiplex PCR and multi-color fluorescent in situ hybridization ( m -FISH) coupled protocol for detection and imaging of multi-pathogens involved in inflammatory bowel disease

doi: 10.1186/s13099-018-0278-1

Figure Lengend Snippet: Validation of multiplex PCR using individual and combined oligonucleotide primer sets and DNA extracts from IBD tissue. Multiplex PCR was performed on DNA extracts from intestinal tissue samples. DNA template was extracted using the modified DNAzol ® . RS1: ulcerative colitis (UC) patient; RS2: Crohn’s disease (CD) patient; (1) Non-pathogenic E. coli strain K-12 18s primers were used (171 bp); (2) MAP UCF4 IS900 AV1/AV2 primers were used (298 bp); (3) AIEC strain LF82 g ipA primers were used (357 bp); (4) K. pneumoniae 23s primers were used (493 bp); (5) Mycobacterium avium complex (MAC) IS1311 primers were used (534 bp); (6) a cocktail of the 5 primer sets mentioned above were used. M: DNA molecular weight marker

Article Snippet: Non - pathogenic Escherichia coli Strain K-12 , ATCC 8739.

Techniques: Biomarker Discovery, Multiplex Assay, Modification, Molecular Weight, Marker

Journal: Gut Pathogens

Article Title: Development of multiplex PCR and multi-color fluorescent in situ hybridization ( m -FISH) coupled protocol for detection and imaging of multi-pathogens involved in inflammatory bowel disease

doi: 10.1186/s13099-018-0278-1

Figure Lengend Snippet: Gram stain, acid-fast stain and m -FISH detection of bacterial cultures. Gram stain (1), acid-fast stain (2), and oligonucleotide m -FISH (3) images of bacterial cultures: a , b Non-pathogenic E. coli strain K-12; c MAP UCF4; and d K. pneumoniae . For - m -FISH detection: a 3 m -FISH using non-pathogenic E. coli K-12 18s probe labeled with AF647 fluorophore; b 3 m -FISH using AIEC strain LF82 g ipA probe labeled with AF568 fluorophore; c 3 m -FISH using MAP UCF4 IS900 AV1 probe labeled with AF488 fluorophore; and d 3 m-FISH using K. pneumoniae 23s probe labeled with AF546 fluorophore. All microscopic images were obtained at 1000 × magnification. White measurement bar found in D3 represents 100 μm. All m -FISH images were obtained using CSLM

Article Snippet: Non - pathogenic Escherichia coli Strain K-12 , ATCC 8739.

Techniques: Staining, Ziehl-Neelsen Stain, Labeling

Journal: Gut Pathogens

Article Title: Development of multiplex PCR and multi-color fluorescent in situ hybridization ( m -FISH) coupled protocol for detection and imaging of multi-pathogens involved in inflammatory bowel disease

doi: 10.1186/s13099-018-0278-1

Figure Lengend Snippet: Use of m -FISH using individual probes to detect multiple pathogens in intestinal tissue. Oligonucleotide m -FISH analysis in tissue sections from patients with UC (RS1) and CD (RS2) was done targeting for: a Non-pathogenic E. coli strain K-12; b AIEC strain LF82; c MAP; and d K. pneumoniae . Images illustrating DAPI are in red. The individual m -FISH probes that were used were: a non-pathogenic E. coli strain K-12 18s probe labeled with AF647 fluorophore; b AIEC strain LF82 g ipA probe labeled with AF568 fluorophore; c MAP UCF4 IS900 AV1 probe labeled with AF488 fluorophore; and d K. pneumoniae 23s probe labeled with AF546 fluorophore. White measurement bars found in D1 and D2 represents 20 μm. All m -FISH images were obtained using CSLM

Article Snippet: Non - pathogenic Escherichia coli Strain K-12 , ATCC 8739.

Techniques: Labeling

Journal: Gut Pathogens

Article Title: Development of multiplex PCR and multi-color fluorescent in situ hybridization ( m -FISH) coupled protocol for detection and imaging of multi-pathogens involved in inflammatory bowel disease

doi: 10.1186/s13099-018-0278-1

Figure Lengend Snippet: Use of m -FISH using combined probes to detect multiple pathogens in intestinal tissue from CD. Oligonucleotide m -FISH images of intestinal tissue sections from four CD patients ( a RS3, b RS4, c RS5, and d RS6). A combined probes mixture including (2) EC647 (non-pathogenic E. coli strain K-12 18s probe labeled with AF647 fluorophore) and (3) MAP488 (MAP UCF4 IS900 AV1 probe labeled with AF488 fluorophore) were used in all tissue sections. (1) DAPI staining and (4) merged images between DAPI and the corresponding probe. White measurement bar located in B3 represents 20 μm. All m -FISH images were obtained using CSLM

Article Snippet: Non - pathogenic Escherichia coli Strain K-12 , ATCC 8739.

Techniques: Labeling, Staining

Journal: Gut Pathogens

Article Title: Development of multiplex PCR and multi-color fluorescent in situ hybridization ( m -FISH) coupled protocol for detection and imaging of multi-pathogens involved in inflammatory bowel disease

doi: 10.1186/s13099-018-0278-1

Figure Lengend Snippet: Nucleotide primers used in multiplex PCR and m -FISH probes

Article Snippet: Non - pathogenic Escherichia coli Strain K-12 , ATCC 8739.

Techniques: Multiplex Assay

Journal: Gut Pathogens

Article Title: Development of multiplex PCR and multi-color fluorescent in situ hybridization ( m -FISH) coupled protocol for detection and imaging of multi-pathogens involved in inflammatory bowel disease

doi: 10.1186/s13099-018-0278-1

Figure Lengend Snippet: Microorganisms used in this study

Article Snippet: Non - pathogenic Escherichia coli Strain K-12 , ATCC 8739.

Techniques:

Journal: Infection and Drug Resistance

Article Title: Plasmid Profiling and Occurrence of β-Lactamase Enzymes in Multidrug-Resistant Uropathogenic Escherichia coli in Kathmandu, Nepal

doi: 10.2147/IDR.S250591

Figure Lengend Snippet: Antibiotic Susceptibility Pattern of MDR E. coli Isolates (n=170)

Article Snippet: The reference strains, ESBLs producing E. coli NCTC 13351, non-ESBLs producing E. coli ATCC 25922, MBLs producing E. coli NCTC 13476 and non-MBLs producing E. coli ATCC 25922 from Institue of Medicine, Tribhuvan University Teaching Hospital, were included as controls in the study.

Techniques:

Journal: Infection and Drug Resistance

Article Title: Plasmid Profiling and Occurrence of β-Lactamase Enzymes in Multidrug-Resistant Uropathogenic Escherichia coli in Kathmandu, Nepal

doi: 10.2147/IDR.S250591

Figure Lengend Snippet: Antibiotic Resistance Pattern of Clinical Isolates of MDR E. coli Isolates

Article Snippet: The reference strains, ESBLs producing E. coli NCTC 13351, non-ESBLs producing E. coli ATCC 25922, MBLs producing E. coli NCTC 13476 and non-MBLs producing E. coli ATCC 25922 from Institue of Medicine, Tribhuvan University Teaching Hospital, were included as controls in the study.

Techniques:

Journal: Infection and Drug Resistance

Article Title: Plasmid Profiling and Occurrence of β-Lactamase Enzymes in Multidrug-Resistant Uropathogenic Escherichia coli in Kathmandu, Nepal

doi: 10.2147/IDR.S250591

Figure Lengend Snippet: Distribution of different β-lactamases among MDR E. coli isolates.

Article Snippet: The reference strains, ESBLs producing E. coli NCTC 13351, non-ESBLs producing E. coli ATCC 25922, MBLs producing E. coli NCTC 13476 and non-MBLs producing E. coli ATCC 25922 from Institue of Medicine, Tribhuvan University Teaching Hospital, were included as controls in the study.

Techniques:

Journal: Infection and Drug Resistance

Article Title: Plasmid Profiling and Occurrence of β-Lactamase Enzymes in Multidrug-Resistant Uropathogenic Escherichia coli in Kathmandu, Nepal

doi: 10.2147/IDR.S250591

Figure Lengend Snippet: Plasmid Profile of MDR E. coli Isolates

Article Snippet: The reference strains, ESBLs producing E. coli NCTC 13351, non-ESBLs producing E. coli ATCC 25922, MBLs producing E. coli NCTC 13476 and non-MBLs producing E. coli ATCC 25922 from Institue of Medicine, Tribhuvan University Teaching Hospital, were included as controls in the study.

Techniques: Plasmid Preparation, Molecular Weight

Journal: Infection and Drug Resistance

Article Title: Plasmid Profiling and Occurrence of β-Lactamase Enzymes in Multidrug-Resistant Uropathogenic Escherichia coli in Kathmandu, Nepal

doi: 10.2147/IDR.S250591

Figure Lengend Snippet: Plasmid Profiling in Relation to β-Lactamase Production in MDR E. coli Isolates

Article Snippet: The reference strains, ESBLs producing E. coli NCTC 13351, non-ESBLs producing E. coli ATCC 25922, MBLs producing E. coli NCTC 13476 and non-MBLs producing E. coli ATCC 25922 from Institue of Medicine, Tribhuvan University Teaching Hospital, were included as controls in the study.

Techniques: Plasmid Preparation, Molecular Weight

Journal: Infection and Drug Resistance

Article Title: Plasmid Profiling and Occurrence of β-Lactamase Enzymes in Multidrug-Resistant Uropathogenic Escherichia coli in Kathmandu, Nepal

doi: 10.2147/IDR.S250591

Figure Lengend Snippet: Separation of plasmid DNA molecular weight on agarose gel stained with ethidium bromide (lanes 1–16: plasmid DNA of clinical isolates of MDR E. coli , lane (M) marker DNA – 33.5 kb DNA ladder).

Article Snippet: The reference strains, ESBLs producing E. coli NCTC 13351, non-ESBLs producing E. coli ATCC 25922, MBLs producing E. coli NCTC 13476 and non-MBLs producing E. coli ATCC 25922 from Institue of Medicine, Tribhuvan University Teaching Hospital, were included as controls in the study.

Techniques: Plasmid Preparation, Molecular Weight, Agarose Gel Electrophoresis, Staining, Marker

Journal: Nutrients

Article Title: Macrofungal Extracts as a Source of Bioactive Compounds for Cosmetical Anti-Aging Therapy: A Comprehensive Review

doi: 10.3390/nu16162810

Figure Lengend Snippet: Overview of research on the anti-aging effects of mushroom extracts on skin.

Article Snippet: , in vitro Mus musculus skin melanoma cell line (B16-F10 ATCC, CRL-6475) and Cercopithecus aethiops kidney normal cell line (Vero ATCC, CCL-81); mouse fibroblast cell line (L929) , The multifunctional peptide derived from Ganoderma lucidum was sequenced via LC-MS/MS as NH2-PVRSSNCA-CO2H (octapeptide). Its antioxidant activity was evaluated at 1 mg/mL using DPPH, ABTS and FRAP assays. The octapeptide demonstrated antioxidant capacities of 0.121 ± 0.01 mg ascorbic acid equivalent, 0.173 ± 0.03 mg gallic acid equivalent and 2.21 ± 0.23 mM FeSO 4 equivalent, comparable to established antioxidants. Proteomics analysis identified 5804 proteins and highlighted several pathways affected by the octapeptide in melanoma cells. Targeted proteomics revealed that pigmentation-related proteins were upregulated, while Tyrosinase-Related Protein 1 was downregulated in the treated group. The octapeptide, at concentrations from 1.5625 μg/mL to 100 μg/mL, did not cause significant cell death in either melanoma or Vero cells. , [ ] .

Techniques: In Vitro, Expressing, Functional Assay, Modification, Concentration Assay, Inhibition, Antioxidant Activity Assay, In Vivo, Extraction, Ethanol Precipitation, Emulsion, Activity Assay, Purification, Molecular Weight, Mouse Assay, FRAP Assay, Positive Control, Isolation, Irradiation, Activation Assay, Protein-Protein interactions, Derivative Assay, Targeted Proteomics, In Silico, Cell Cycle Assay, Phospho-proteomics, Produced, Control, Generated, Cream, Formulation, Immunohistochemical staining, Sonication, Negative Control, Diffusion-based Assay, Zeta Potential Analyzer

Journal: PLoS ONE

Article Title: A Rapid Subtractive Immunization Method to Prepare Discriminatory Monoclonal Antibodies for Food E. coli O157:H7 Contamination

doi: 10.1371/journal.pone.0031352

Figure Lengend Snippet: Bacterial strains used in the study.

Article Snippet: The following strains were tested including Salmonella sp. (CMCC 50325), Citrobacter freundii (IHEM 1.5001), Staphylococcus aureus (CMCC 29213), Shigella (CMCC 51081,51066), Non-O157 E. coli (CMCC 44216, 44505), E. coli O157:H19 (CMCC 44752), E. coli O157:H7 (ATCC 43895).

Techniques:

Journal: PLoS ONE

Article Title: A Rapid Subtractive Immunization Method to Prepare Discriminatory Monoclonal Antibodies for Food E. coli O157:H7 Contamination

doi: 10.1371/journal.pone.0031352

Figure Lengend Snippet: A, sera from Cy-treated mice (experimental group) and no Cy-treated mice (control group) were diluted and tested for antibody reactivity to E. coli O157:H19 in whole cell ELISA (OD 450 nm); B, sera were diluted and tested for reactivity to E. coli O157:H7 and E. coli O157:H19 (▪: experimental group; •: control group).

Article Snippet: The following strains were tested including Salmonella sp. (CMCC 50325), Citrobacter freundii (IHEM 1.5001), Staphylococcus aureus (CMCC 29213), Shigella (CMCC 51081,51066), Non-O157 E. coli (CMCC 44216, 44505), E. coli O157:H19 (CMCC 44752), E. coli O157:H7 (ATCC 43895).

Techniques: Control, Enzyme-linked Immunosorbent Assay

Journal: PLoS ONE

Article Title: A Rapid Subtractive Immunization Method to Prepare Discriminatory Monoclonal Antibodies for Food E. coli O157:H7 Contamination

doi: 10.1371/journal.pone.0031352

Figure Lengend Snippet: Summary of the characteristic of some mAbs reacting with Escherichia coli O157:H7 and others.

Article Snippet: The following strains were tested including Salmonella sp. (CMCC 50325), Citrobacter freundii (IHEM 1.5001), Staphylococcus aureus (CMCC 29213), Shigella (CMCC 51081,51066), Non-O157 E. coli (CMCC 44216, 44505), E. coli O157:H19 (CMCC 44752), E. coli O157:H7 (ATCC 43895).

Techniques:

Journal: PLoS ONE

Article Title: A Rapid Subtractive Immunization Method to Prepare Discriminatory Monoclonal Antibodies for Food E. coli O157:H7 Contamination

doi: 10.1371/journal.pone.0031352

Figure Lengend Snippet: The characteristics of mAbs against E. coli O157:H7.

Article Snippet: The following strains were tested including Salmonella sp. (CMCC 50325), Citrobacter freundii (IHEM 1.5001), Staphylococcus aureus (CMCC 29213), Shigella (CMCC 51081,51066), Non-O157 E. coli (CMCC 44216, 44505), E. coli O157:H19 (CMCC 44752), E. coli O157:H7 (ATCC 43895).

Techniques:

Journal: PLoS ONE

Article Title: A Rapid Subtractive Immunization Method to Prepare Discriminatory Monoclonal Antibodies for Food E. coli O157:H7 Contamination

doi: 10.1371/journal.pone.0031352

Figure Lengend Snippet: E. coli O157:H7 cell lysates were subjected to SDS–PAGE, transferred to nitrocellulose filter, and probed with 1C6 (lane 1), 1D8, (lane 2), 4A7 (lane 3), 5A2 (lane 4), 5D8 (lane 5). The sizes of molecular weight standards are shown at the right (lane 6).

Article Snippet: The following strains were tested including Salmonella sp. (CMCC 50325), Citrobacter freundii (IHEM 1.5001), Staphylococcus aureus (CMCC 29213), Shigella (CMCC 51081,51066), Non-O157 E. coli (CMCC 44216, 44505), E. coli O157:H19 (CMCC 44752), E. coli O157:H7 (ATCC 43895).

Techniques: SDS Page, Molecular Weight

Journal: PLoS ONE

Article Title: A Rapid Subtractive Immunization Method to Prepare Discriminatory Monoclonal Antibodies for Food E. coli O157:H7 Contamination

doi: 10.1371/journal.pone.0031352

Figure Lengend Snippet: Immune-colloidal gold probes coated with mAb 5A2 were applied to special single out E. coli O157:H7 contaminated minced beef from others which included food samples contaminated by ATCC 43895 (A1,B1,C1,D1), IHEM 1.3001 (A2), IHEM 1.3002 (A3), IHEM 1.3003 (A4), CMCC 50303 (B2), CMCC 50115 (B3), CMCC 50309 (B4), CMCC 51135 (C2), CMCC 51081 (C3), CMCC 51066 (C4), CMCC 44102 (D2), CMCC 44109 (D3), CMCC 44156 (D4). Negative control A5, B5, C5, D5) refers to food samples which have no bacterial contamination.

Article Snippet: The following strains were tested including Salmonella sp. (CMCC 50325), Citrobacter freundii (IHEM 1.5001), Staphylococcus aureus (CMCC 29213), Shigella (CMCC 51081,51066), Non-O157 E. coli (CMCC 44216, 44505), E. coli O157:H19 (CMCC 44752), E. coli O157:H7 (ATCC 43895).

Techniques: Negative Control

Journal: Cancer cell

Article Title: A Platform of Synthetic Lethal Gene Interaction Networks Reveals that the GNAQ Uveal Melanoma Oncogene Controls the Hippo Pathway through FAK

doi: 10.1016/j.ccell.2019.01.009

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies YAP Cell Signaling Technology, MA 14074 pS127-YAP Cell Signaling Technology, MA 4911 pS909-LATS1 Cell Signaling Technology, MA 9157 pT1079-LATS1 Cell Signaling Technology, MA 8654 LATS1 Cell Signaling Technology, MA 3477 P-MST1/MST2 Cell Signaling Technology, MA 3681 MST1 Cell Signaling Technology, MA 3682 GAPDH(14C10) Cell Signaling Technology, MA 2118 α-Tubulin Cell Signaling Technology, MA 3873 pY Cell Signaling Technology, MA 9411 HA-tag-HRP Cell Signaling Technology, MA 2999 HA-tag Cell Signaling Technology, MA 3724 myc-tag Cell Signaling Technology, MA 2278 pY397-FAK Cell Signaling Technology, MA 8556 FAK Cell Signaling Technology, MA 3285 cleaved PARP Cell Signaling Technology, MA 9541 p-ERK1/2 Cell Signaling Technology, MA 4370 ERK1/2 Cell Signaling Technology, MA 4696 MOB1 Cell Signaling Technology, MA 13730 pT35-MOB1 Cell Signaling Technology, MA 8699 Gαq(E-17) Santa Cruz Biotech., CA sc-393 FAK(C-20) Santa Cruz Biotech., CA sc-558 RhoA Cell Signaling Technology, MA 2117 TRIO(H120) Santa Cruz Biotech., CA sc-28564 Rac1 BD Biosciences, CA 610651 pY357-YAP Abcam, MA ab62751 LATS2 Bethyl Laboratories, TX A300–479A pY26-MOB1A Signalway Antibody, MA 12878 flag-tag-HRP Sigma-Aldrich, MO A8592 Ki67 DAKO, CA M724029–2 Bacterial strains DH5alpha Competent E. coli BioPioneer, CA GACC-96 Stbl3 Competent E. coli Thermo Fisher C737303 siRNAs Non-targeting Dharmacon, CO D-001810–0X GNAQ Sigma-Aldrich, MO SASI Hs01 00231793 PTK2 Thermo Fisher, MA s11485 AKT1 Thermo Fisher, MA s659 MGLL Thermo Fisher, MA s22380 MTHFD1 Thermo Fisher, MA s9032 CDK1 Thermo Fisher, MA s464 SIRT1 Thermo Fisher, MA s223591 PSMB5 Thermo Fisher, MA s11354 TRIO Dharmacon, CO L-005047–00-0005 RHOA Dharmacon, CO L-003860–00-0005 RAC1 Dharmacon, CO L-003560–00-0005 LATS1 Sigma-Aldrich, MO Hs01 00046128 LATS2 Sigma-Aldrich, MO Hs01 00158803 DNAs pCMV-myc-MST1 Addgene 8847 pCMV2-FLAG-SAV1 Addgene 18970 pcDNA3-HA-MOB1 Addgene 32835 pcDNA3-HA-Y26F-MOB1 Generated in-lab NA pLENTi-HA-MOB1 Generated in-lab NA pLENTi-HA-Y26F-MOB1 Generated in-lab NA pGEX-HA-MOB1 Generated in-lab NA pGEX-HA-Y26F-MOB1 Generated in-lab NA pLVX-T etOne-FLAG-FAK Generated in-lab NA p2xFLAG-CMV2-LATS1 Addgene 18971 8xGTIIC-luciferase Addgene 34615 REAGENT alamarBlueTM Reagent Grand Island, NY DAL1100 FAK Kinase Enzyme System Promega V1971 YAP1 (Human) Recombinant Abnova H00010413-P01 Protein Glutathione Sepharose 4B GE Healthcare 17–0756-01 N/C Extraction Reagents ThermoFisher 78833 Software and Algorithms ISLE Lee et al 2018 https://www.github.com/jooslee/ISLE Open in a separate window KEY RESOURCES TABLE Despite the central role of Hippo/YAP-regulating mechanisms in uveal melanoma (UM), there are no clinically effective therapeutic targets.

Techniques: Generated, Recombinant, Extraction, Software

Journal: bioRxiv

Article Title: Centimeter-scale quorum sensing dictates collective survival of differentiating embryonic stem cells

doi: 10.1101/2020.12.20.423651

Figure Lengend Snippet: ( A ) Main graph: fold-change in initial population density, after 6 days in N2B27 supplemented with any of the 11 recombinant molecules (indicated on horizontal axis) or all of 11 combined (“All”) at day 0. All data are for initially low-density population (862 cells / cm 2 ). Data for 46C cells differentiating in N2B27+RA that were previously self-renewing in serum with LIF. Also see STAR Methods and for full details of concentrations added. Horizontal dashed line shows the maximum fold-change in density obtained in our study (nearly 4 fold), which occurs when the same low-density population is rescued by a high-density population’s (5172 cells / cm 2 initially) medium. Middle inset: fold-change in initial population density after adding various concentrations of recombinant FGF4 (horizontal axis). Same procedure as described for main graph. Right inset: foldchange in initial population density (black bar) and percentage of cells entering NE lineage (Sox1-GFP expressing cells) (green bar), both measured 10 days after differentiation begins in presence of 200 ngImL FGF4 that we added at the start of differentiation. Error bars are s.e.m.; n = 3. ( B ) ELISA measurements of concentrations of extracellular FGF4 in the medium of a high-density population (8620 cells / cm 2 initially) during unguided differentiation in N2B27 during 2 days (blue points) (previously self-renewing in serum with LIF) (see STAR Methods). Vertical axis shows FGF4 concentration relative to that of a ~80% confluent pluripotent population (denoted “1x” and marked with yellow horizontal line). 80% confluency equals i~8 x 10 6 cells in 10-cm diameter dish. Lower detection limit of the ELISA assay is indicated (in grey). See for ELISA standard curves. Error bars are s.e.m.; n = 3. ( C ) Cartoon shows PD173074, a well-characterized smallmolecule inhibitor of FGF receptors ( , ) (see STAR Methods). Fold-change in initial population density (bottom graph) and percentage of populations that enter NE lineage (top graph), both measured 6 days after differentiation began and as a function of initial population density. Data for 46C cells differentiating in N2B27+RA that were previously self-renewing in serum with LIF. Red data points in both graphs are for populations that were incubated with 2 μM (1056 ngImL) of PD173074 from the start of differentiation. PD173074 was dissolved in DMSO. Thus, as a control, black and green points are for populations without PD173074 but with the same amount of DMSO (volume per volume) as the populations represented by red data points. Blue shade indicates population expansion and red shade indicates population extinction. Error bars are s.e.m.; n = 3. ( D ) Heat map showing transcriptome-wide changes in unguided differentiation (N2B27) of 46C cells (previously self-renewing in serum with LIF) of low-density population (862 cells / cm 2 ; enclosed in pink box), near-threshold (medium-density) population (1931 cells / cm 2 ; enclosed in grey box), and high-density population (5172 cells / cm 2 ; enclosed in blue box) (see STAR Methods). Leftmost column shows data for self-renewal (pluripotent) population before differentiation begins (labeled “All” since every population starts as this population before differentiation). Each column of differentiating population shows data for 1 day after (labeled “1”) or 2 days after (labeled “2”) starting differentiation. Each row shows a different gene, each of which are either activated (21 genes) or repressed (19 genes) by YAP1, either directly or indirectly. lists all genes. Color represents row Z-score: a measure of by how much a gene’s expression level for a given condition deviates from that gene’s expression level averaged across all different conditions (i.e., different populations and days). Purple represents a positive row Z-score (more expressed than average). Orange represents a negative row Z-score (less expressed than average). Data based on 3 biological replicates. ( E ) Cartoon shows YAP1 which exists as either phosphorylated (labeled “P”) or dephosphorylated. Verteporfin (VP) inhibits active (dephosphorylated) YAP1 from entering the nucleus and regulating target gene expression. Fold-change in population density for high-density population (5172 cells / cm 2 initially, in blue box) and low-density population that was rescued with medium of 2-days-old high-density population (862 cells / cm 2 initially, in green box) after 6 days of differentiation towards NE-lineage. Data for 46C cells differentiating in N2B27+RA that were previously self-renewing in serum with LIF. Black bar: Verteporfin (VP) was always absent. Red bar in middle of each box: VP was added to medium after the first two days. Third column of each box shows absence of cells (extinction) when VP was present from the start of differentiation. Also see for full data. Error bars are s.e.m.; n = 3. ( F ) ELISA measurements showing amounts of YAP1 protein phosphorylated at Ser397 (inactive YAP1) (see STAR Methods and also ). Vertical axis shows the relative amount of inactive YAP1: the level of inactive YAP1 for a differentiating population divided by the amount of inactive YAP1 for a pluripotent population (in serum with LIF medium) that has the same cell numbers as the differentiating population at the time of lysing the cells for ELISA. Values are for 46C cells previously propagated in serum with LIF medium, 3 days after starting differentiation towards NE lineage with N2B27+RA medium. Pink: low-density population (862 cells / cm 2 initially). Blue: high-density population (5172 cells / cm 2 initially). Grey: low-density population rescued after two days by medium from a 2-day-old high-density population. Green: low-density population rescued by adding 200 ngImL FGF4 to its medium on day 0 (see (A)). Error bars are s.e.m.; n = 3. ( G ) Bcl2 (anti-apoptotic gene) and Cyr61 (YAP1-specific target) expression levels over time after initiating differentiation. Data obtained with RT-qPCR for 46C cells differentiating in N2B27+RA that were previously self-renewing in serum with LIF (see STAR Methods). Same color scheme as shown in (F). Also see for other genes. On each day, we first normalized a population’s gene ( Bcl2 or Cyr61 ) expression level by that population’s Gapdh level (housekeeping gene). Afterwards, plotted on the vertical axis, we divided each population’s Gapdh -normalized gene ( Bcl2 and Cyr61 ) expression level on a given day by the Gapdh -normalized value for one-day-old low-density population (whose value is thus “1x” here). Error bars are s.e.m.; n = 3.

Article Snippet: After 2 days of culturing in N2B27, we added 500 nM of Retinoic Acid, and one or combinations of the following recombinant proteins to the medium: 200 ng/mL of recombinant mouse FGF4 (R&D Systems, #7486-F4), 200 ng/mL of recombinant human FGF5 (R&D Systems, #237-F5), 100 ng/mL of recombinant mouse PDGFA (Novus, NBP1-43148), 100 ng/mL of recombinant mouse VEGFB 186 (Novus, #767-VE), 100 ng/mL of recombinant mouse VEGFA (Novus, #493-MV), 500 ng/mL of recombinant human CYR61/CCN1 (Novus, #4055-CR), 500 ng/mL of recombinant human CTGF/CCN2 (Novus, #9190-CC), 200 ng/mL of recombinant mouse CLU (Novus, #2747-HS), 500 ng/mL of recombinant human HSPA8/HSC70 (Novus, #NBP1-30278), 1000 ng/mL of recombinant human Cyclophilin A (PPIA) (Novus, #NBC1-18425), or 2000 ng/mL of mouse recombinant SCF (STEMCELL, #78064).

Techniques: Recombinant, Expressing, Enzyme-linked Immunosorbent Assay, Concentration Assay, Incubation, Control, Labeling, Targeted Gene Expression, Quantitative RT-PCR

Journal: bioRxiv

Article Title: Centimeter-scale quorum sensing dictates collective survival of differentiating embryonic stem cells

doi: 10.1101/2020.12.20.423651

Figure Lengend Snippet: ( A ) Spreading a relatively small number of cells (~5000 cells / mL of N2B27) on a 10-cm diameter dish yields small colonies (size ~ 400 μm 2 ) at the start of differentiation which eventually become extinct over time. Images are for 46C cells differentiating in N2B27+RA that were previously self-renewing in serum with LIF. Scale bars = 200 μm. Microscopy images are taken after 24 hours and 120 hours. ( B ) A same number of cells as in shown in (A) confined within a small area with a droplet on a 10-cm diameter dish yields macroscopic colony (size ~ 28 mm 2 ) at the start of differentiation which eventually survives, expands and differentiates over time. Images are for 46C cells differentiating in N2B27+RA that were previously self-renewing in serum with LIF. Scale bar = 2 mm. Microscopy images are taken after 24 hours and 120 hours. In each of the two images, we stitched together multiple fields of view, with each field of view being 1.40 mm x 0.99 mm (same field size as in ). The stitching creates the checkered outline in both images. ( C ) Macroscopic colony, according to procedure described in (B), is visible to the naked eye after several days of differentiation (shown here is after 10 days of differentiation in N2B27+RA). Image taken after 10 days. In the greyscale image at the bottom, we stitched together multiple fields of view, with each field of view being 1.40 mm x 0.99 mm (same field size as in ). The stitching creates the checkered outline in this image. ( D ) Summary of strategies for inducing collective survival of differentiating ES cells. Spreading a relatively low number of cells (5000 cells / mL of N2B27) on a 10-cm diameter dish (shown here: 58 cm 2 surface area (862 cells / cm 2 ) and 10-mL N2B27) results in all cells becoming extinct during differentiation. Adding recombinant FGF4 (200 ngImL) to supernatant rescues spread cells from extinction (~1.3 fold-growth and ~19% Sox1-GFP positive). Lowering the height of liquid medium over millimetres rescues spread cells from extinction (~2.9 fold-growth and ~71% Sox1-GFP positive). Transplanting spread cells into a high-density population’s medium on day 2 rescues cells from extinction (~4.3 fold-growth and ~39% Sox1-GFP positive). Clustering a same number of cells, also used for spreading, into a macroscopic colony at the start of differentiation rescues cells from extinction (~20 fold-growth and ~92% Sox1-GFP positive). Data for 46C cells differentiating in N2B27+RA that were previously self-renewing in serum with LIF.

Article Snippet: After 2 days of culturing in N2B27, we added 500 nM of Retinoic Acid, and one or combinations of the following recombinant proteins to the medium: 200 ng/mL of recombinant mouse FGF4 (R&D Systems, #7486-F4), 200 ng/mL of recombinant human FGF5 (R&D Systems, #237-F5), 100 ng/mL of recombinant mouse PDGFA (Novus, NBP1-43148), 100 ng/mL of recombinant mouse VEGFB 186 (Novus, #767-VE), 100 ng/mL of recombinant mouse VEGFA (Novus, #493-MV), 500 ng/mL of recombinant human CYR61/CCN1 (Novus, #4055-CR), 500 ng/mL of recombinant human CTGF/CCN2 (Novus, #9190-CC), 200 ng/mL of recombinant mouse CLU (Novus, #2747-HS), 500 ng/mL of recombinant human HSPA8/HSC70 (Novus, #NBP1-30278), 1000 ng/mL of recombinant human Cyclophilin A (PPIA) (Novus, #NBC1-18425), or 2000 ng/mL of mouse recombinant SCF (STEMCELL, #78064).

Techniques: Microscopy, Recombinant

Journal: bioRxiv

Article Title: Centimeter-scale quorum sensing dictates collective survival of differentiating embryonic stem cells

doi: 10.1101/2020.12.20.423651

Figure Lengend Snippet: Data for 46C cells (which have Sox1 promoter driving GFP expression) differentiating towards NE lineage in N2B27 (without any inducers such as RA) that were previously self-renewing in serum+LIF (see STAR Methods). Same RNA-Seq dataset as in . To identify secreted factors other than FGF4 that might contribute to determining a population survival, we performed RNA-Seq to detect expression of any secreted factors that are known to control cell proliferation and/or death. We performed RNA-Seq on four populations: (1) pluripotent population prior to differentiation; (2) low-density (862 cells/cm 2 ) population; (3) high-density (5172 cells/cm 2 ) population; and (4) medium-density (1931 cells/cm 2 ) population that is near the threshold density. For the three differentiating populations, we collected their cells on the first and second day after triggering differentiation. Expression levels (FPKMs) of secreted factors that are known to control proliferation and/or apoptosis in ES cells and that fall within the range of molecular weights that the membranefilter experiments identified (50 – 300 kDa with +/-50% error) . Shown are the following genes: Ctgf, Scf, Ppia, Clu, Vegfa, Vegfb, Cyr61, Fgf5, Pdgfa, Fgf4 and Hspa8 . Below each gene name is the molecule’s weight (kDa) according to two online resources: Uniprot and ExPASy. n = 3 for all plots; Error bars are s.e.m.

Article Snippet: After 2 days of culturing in N2B27, we added 500 nM of Retinoic Acid, and one or combinations of the following recombinant proteins to the medium: 200 ng/mL of recombinant mouse FGF4 (R&D Systems, #7486-F4), 200 ng/mL of recombinant human FGF5 (R&D Systems, #237-F5), 100 ng/mL of recombinant mouse PDGFA (Novus, NBP1-43148), 100 ng/mL of recombinant mouse VEGFB 186 (Novus, #767-VE), 100 ng/mL of recombinant mouse VEGFA (Novus, #493-MV), 500 ng/mL of recombinant human CYR61/CCN1 (Novus, #4055-CR), 500 ng/mL of recombinant human CTGF/CCN2 (Novus, #9190-CC), 200 ng/mL of recombinant mouse CLU (Novus, #2747-HS), 500 ng/mL of recombinant human HSPA8/HSC70 (Novus, #NBP1-30278), 1000 ng/mL of recombinant human Cyclophilin A (PPIA) (Novus, #NBC1-18425), or 2000 ng/mL of mouse recombinant SCF (STEMCELL, #78064).

Techniques: Expressing, RNA Sequencing, Control

Journal: bioRxiv

Article Title: Centimeter-scale quorum sensing dictates collective survival of differentiating embryonic stem cells

doi: 10.1101/2020.12.20.423651

Figure Lengend Snippet: Data for 46C cells (which have Sox1 promoter driving GFP expression) differentiating towards NE lineage in N2B27 (without any inducers such as RA) that were previously selfrenewing in serum+LIF (see STAR Methods). Same RNA-Seq dataset as in . We performed RNA-Seq to measure the expression levels of all 22 FGF ligands (A) and their receptors (FGFRs) (B). We performed RNA-Seq on four populations: (1) pluripotent population prior to differentiation; (2) low-density (862 cells/cm 2 ) population; (3) high-density (5172 cells/cm 2 ) population; and (4) medium-density (1931 cells/cm 2 ) population that is near the threshold density. For the three differentiating populations, we collected their cells on the first and second day after triggering differentiation. (A) Expression levels of all FGF ligands. Shown are the following genes: Fgf1-8, Fgf20-21 and Fgf23 . Below each gene name is the corresponding molecular weight in kDa. Note that FGF4 expression prominently stands out among all the FGFs. n = 3 for all plots; Error bars are s.e.m. (B) Expression levels of all FGF receptors (FGFRs). Shown are the following genes: Fgfr1-4 . Below each gene name is the corresponding molecular weight in kDa, according to two online resources: Uniprot and ExPASy. n = 3 for all plots; Error bars are s.e.m. (C) Expression levels of Wnt ligands. Shown are the following genes: Wnt6, Wnt10a, Wnt9a, Wnt3, Wnt3a, Wnt9b, Wnt5a, Wnt1, Wnt8a, Wnt8b, Wnt2b, Wnt4, Wnt16, Wnt7a, Wnt5b and Wnt11 . None of the Wnt genes prominently stand out, except for Wnt4 which still has an order of magnitude lower expression relative to FGF4 expression. n = 3 for all plots; Error bars are s.e.m.

Article Snippet: After 2 days of culturing in N2B27, we added 500 nM of Retinoic Acid, and one or combinations of the following recombinant proteins to the medium: 200 ng/mL of recombinant mouse FGF4 (R&D Systems, #7486-F4), 200 ng/mL of recombinant human FGF5 (R&D Systems, #237-F5), 100 ng/mL of recombinant mouse PDGFA (Novus, NBP1-43148), 100 ng/mL of recombinant mouse VEGFB 186 (Novus, #767-VE), 100 ng/mL of recombinant mouse VEGFA (Novus, #493-MV), 500 ng/mL of recombinant human CYR61/CCN1 (Novus, #4055-CR), 500 ng/mL of recombinant human CTGF/CCN2 (Novus, #9190-CC), 200 ng/mL of recombinant mouse CLU (Novus, #2747-HS), 500 ng/mL of recombinant human HSPA8/HSC70 (Novus, #NBP1-30278), 1000 ng/mL of recombinant human Cyclophilin A (PPIA) (Novus, #NBC1-18425), or 2000 ng/mL of mouse recombinant SCF (STEMCELL, #78064).

Techniques: Expressing, RNA Sequencing, Molecular Weight

Journal: bioRxiv

Article Title: Centimeter-scale quorum sensing dictates collective survival of differentiating embryonic stem cells

doi: 10.1101/2020.12.20.423651

Figure Lengend Snippet: The RNA-Seq revealed that 11 secreted factors that are known to control cell proliferation/and or death were highly expressed in differentiating, high-density populations. We thus reasoned that one or combinations of these factors may be the secreted molecule(s) that determine the survival-versus-extinction fate of a population. Data in (A) and (B) for 46C cells (which have Sox1 promoter driving GFP expression) differentiating towards NE lineage in N2B27+RA that were previously self-renewing in serum+LIF (see STAR Methods). (A) We tested these molecules by adding them one-by-one into the medium of a low-density population (862 cells/cm 2 ) that would ordinarily become extinct. We added the following molecules individually, each at a saturating concentration (also see STAR Methods): version of recombinant mouse FGF4 used in (200 ng/mL), recombinant human FGF5 (200 ng/mL), recombinant mouse PDGFA (100 ng/mL), recombinant mouse VEGFB 186 (100 ng/mL), recombinant mouse VEGFA (100 ng/mL), recombinant human CYR61 (500 ng/mL), recombinant human CTGF (500 ng/mL), recombinant mouse CLU (200 ng/mL), recombinant human HSPA8 (500 ng/mL), recombinant human CYPA (1000 ng/mL), and recombinant mouse SCF (2000 ng/mL). After 6 days in a medium containing one of these molecules, we measured the fold-change in density (black bars) and differentiation efficiency (green bars) of the low-density population. n = 3; error bars are s.e.m. These results show that only the recombinant mouse FGF4 causes the fold-change in population density to be higher than one. All the other factors resulted in the low-density population either approaching extinction (fold change much less than 1) or becoming extinct (indicated with an asterisk). The black dashed line marks the maximum fold-change in population density achieved when the low-density population grows in the medium of a high-density population. The green dashed line marks the maximum differentiation efficiency achieved when the low-density population grows in the medium of a high-density population. The box beneath the plot shows which signaling factors were mixed together and then given to the low-density population in (B). (B) Results obtained by giving combinations of the 11 factors together to the low-density population, with the ingredients of the mixture indicated in the box below (A). Giving all 11 factors together at once yielded the highest growth (~4-fold increase in population density; black bar), which was virtually identical to the growth obtained with a high-density population’s (5172 cells/cm 2 ) medium (black dashed line). But, with the 11 molecules added together at once, the differentiation efficiency (green) remained rather low at ~20% compared to the ~40% (green dashed line) that we get from incubating the low-density population in the medium of a high-density population. As we progressively reduced the number of signaling factors in the mixture from 11 to 2, we observed only a modest decrease in population growth, down to about ~2 fold. Importantly, recombinant FGF4 was included in all these mixtures.

Article Snippet: After 2 days of culturing in N2B27, we added 500 nM of Retinoic Acid, and one or combinations of the following recombinant proteins to the medium: 200 ng/mL of recombinant mouse FGF4 (R&D Systems, #7486-F4), 200 ng/mL of recombinant human FGF5 (R&D Systems, #237-F5), 100 ng/mL of recombinant mouse PDGFA (Novus, NBP1-43148), 100 ng/mL of recombinant mouse VEGFB 186 (Novus, #767-VE), 100 ng/mL of recombinant mouse VEGFA (Novus, #493-MV), 500 ng/mL of recombinant human CYR61/CCN1 (Novus, #4055-CR), 500 ng/mL of recombinant human CTGF/CCN2 (Novus, #9190-CC), 200 ng/mL of recombinant mouse CLU (Novus, #2747-HS), 500 ng/mL of recombinant human HSPA8/HSC70 (Novus, #NBP1-30278), 1000 ng/mL of recombinant human Cyclophilin A (PPIA) (Novus, #NBC1-18425), or 2000 ng/mL of mouse recombinant SCF (STEMCELL, #78064).

Techniques: RNA Sequencing, Control, Expressing, Concentration Assay, Recombinant

Journal: bioRxiv

Article Title: Centimeter-scale quorum sensing dictates collective survival of differentiating embryonic stem cells

doi: 10.1101/2020.12.20.423651

Figure Lengend Snippet: Data for 46C cells differentiating towards NE lineage in N2B27 (without any inducers such as RA) that were previously self-renewing in serum+LIF (see STAR Methods). We used real-time quantitative PCR (RT-qPCR) to measure the expression levels of all four receptors ( FGFR1-4 ) of Fibroblast Growth Factors (FGFs) and the expression levels of the two FGFs, FGF4 and FGF5 (primers in ). We examined a high-density population (5172 cells/cm 2 ) after two days of differentiation. We normalized the resulting expressions of a gene relative to that of the housekeeping gene, GAPDH of the same population, and then further normalized the resulting value to the pluripotent population’s normalized expression level (similar to the procedure described in the caption for ). Thus, a given gene’s expression level is compared to the pluripotent population’s expression level for that gene. Normalized expression levels of FGF4 and FGF5 (in black) and FGFR1-2 (in red). n = 3; Error bars are s.e.m. Altogether, these results show that FGF4, FGF5 and FGFR1-2 are expressed - and some more so than the pluripotent population (i.e., expression value greater than 1) - during the first 2 days in which ES cells exit pluripotency.

Article Snippet: After 2 days of culturing in N2B27, we added 500 nM of Retinoic Acid, and one or combinations of the following recombinant proteins to the medium: 200 ng/mL of recombinant mouse FGF4 (R&D Systems, #7486-F4), 200 ng/mL of recombinant human FGF5 (R&D Systems, #237-F5), 100 ng/mL of recombinant mouse PDGFA (Novus, NBP1-43148), 100 ng/mL of recombinant mouse VEGFB 186 (Novus, #767-VE), 100 ng/mL of recombinant mouse VEGFA (Novus, #493-MV), 500 ng/mL of recombinant human CYR61/CCN1 (Novus, #4055-CR), 500 ng/mL of recombinant human CTGF/CCN2 (Novus, #9190-CC), 200 ng/mL of recombinant mouse CLU (Novus, #2747-HS), 500 ng/mL of recombinant human HSPA8/HSC70 (Novus, #NBP1-30278), 1000 ng/mL of recombinant human Cyclophilin A (PPIA) (Novus, #NBC1-18425), or 2000 ng/mL of mouse recombinant SCF (STEMCELL, #78064).

Techniques: Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Expressing

Journal: bioRxiv

Article Title: Centimeter-scale quorum sensing dictates collective survival of differentiating embryonic stem cells

doi: 10.1101/2020.12.20.423651

Figure Lengend Snippet: We performed ELISA that detects mouse FGF4 (see STAR Methods). Data for 46C cells differentiating towards NE lineage in N2B27 (without any inducers such as RA) that were previously self-renewing in serum+LIF (see STAR Methods). (A) Standard curve based on a recombinant mouse FGF4 that came with the commercial ELISA kit. Note that this recombinant FGF4 is not necessarily the same version as the FGF4 that our cells secrete. Each measurement (absorbance at 450 nm) was done in duplicate (black data points). Then, we performed a logistic regression on the data by fitting a 4-parameter logistic function (red curve): , where A, B, C and D are constant coefficients and x is the known concentration of the recombinant mouse FGF4 that we added. We found: A = 0.0084, B = 1.313, C = 1312 and D = 6.59. (B) Standard curve based on the version of FGF4 that pluripotent cells secrete into their medium. We first concentrated the medium taken from a highly confluent (~80% confluent) pluripotent population with a 3-kDa filter and then performed ELISA on serially diluted fractions of this concentrated medium. Each measurement (absorbance at 450 nm) was done in duplicate (black points). Then, we performed a logistic regression on the black data points by fitting a 4-parameter logistic function (red curve): , where A, B, C and D as constant coefficients and x as amount of lysed, pluripotent cells (day 0). We found: A = 0.07123, B = 1.184, C = 1.407 and D = 4.069. The standard curve shows that pluripotent ES cells secrete a version of FGF4 that our ELISA can detect. Moreover, it also shows a limitation of our ELISA: the assay can only detect sufficiently high concentration of FGF4 as seen by the fact that it cannot detect any FGF4 in a 1:100 dilution of a concentrated medium taken from a highly confluent ES cells. (C) Standard curve based on recombinant mouse FGF4 from a different manufacturer (not from the ELISA kit) that we could add to the cell-culture medium to rescue low-density populations (also see STAR Methods). Each measurement (absorbance at 450 nm) was done in duplicates (black points). As seen here, the ELISA cannot detect any amounts of this version of FGF4, even when its concentration is 100-folds higher than the highest concentration - of the version supplied by the ELISA kit - that we used in (A). The three standard curves (A-C) show that ELISA is highly sensitive to the form of FGF4 - we used three different forms in each of (A-C). The two versions of FGF4 that are not supplied by the ELISA kit (B-C) are detected with lower efficiency than the version supplied by the kit (A). (D) ELISA measurements of secreted FGF4 (in pg/mL) in various conditions (indicated with labels on the horizontal axis). We detected abundant FGF4 in the pluripotency medium (~500 pg/mL). In the medium of the high-density population (8620 cells/cm 2 ) after two days of differentiation, we detected ~50 pg/mL of FGF4. After 1 day of differentiation, the medium of the high-density population did not contain any detectable amounts of FGF4. Hence, high-density populations take 2 days to accumulate appreciable (detectable) amounts of FGF4.

Article Snippet: After 2 days of culturing in N2B27, we added 500 nM of Retinoic Acid, and one or combinations of the following recombinant proteins to the medium: 200 ng/mL of recombinant mouse FGF4 (R&D Systems, #7486-F4), 200 ng/mL of recombinant human FGF5 (R&D Systems, #237-F5), 100 ng/mL of recombinant mouse PDGFA (Novus, NBP1-43148), 100 ng/mL of recombinant mouse VEGFB 186 (Novus, #767-VE), 100 ng/mL of recombinant mouse VEGFA (Novus, #493-MV), 500 ng/mL of recombinant human CYR61/CCN1 (Novus, #4055-CR), 500 ng/mL of recombinant human CTGF/CCN2 (Novus, #9190-CC), 200 ng/mL of recombinant mouse CLU (Novus, #2747-HS), 500 ng/mL of recombinant human HSPA8/HSC70 (Novus, #NBP1-30278), 1000 ng/mL of recombinant human Cyclophilin A (PPIA) (Novus, #NBC1-18425), or 2000 ng/mL of mouse recombinant SCF (STEMCELL, #78064).

Techniques: Enzyme-linked Immunosorbent Assay, Recombinant, Concentration Assay, Cell Culture

Journal: bioRxiv

Article Title: Centimeter-scale quorum sensing dictates collective survival of differentiating embryonic stem cells

doi: 10.1101/2020.12.20.423651

Figure Lengend Snippet: Determining the degradation rate of FGF4 that cells secrete tells us whether FGF4 can diffuse by millimeters or not, through the Stokes-Einstein equation (see STAR Methods). We performed ELISA that targets FGF4 (see STAR Methods) to determine the concentrations of secreted FGF4 when incubated without cells in liquid medium. For this, we used a 3-kDa filter to concentration the pluripotency medium (serum+LIF) taken from a confluent population of 46C cells. Then we incubated the medium without any cells in a 37°C incubator for the hours indicated on the horizontal axis. We then took it out of the incubator and performed ELISA on it to measure the remaining [FGF4]. We observed that after incubating for 72 hours (3 days), the initial concentration of the secreted FGF4 was not appreciably degraded. As a control, we confirmed that no ingredient of a 3-kDa-concentrated pluripotency medium (without cells) interferes with our ELISA measurement to produce a non-zero concentration. Thus, this control showed no such signal (see ). Altogether, this result suggests that the concentration of secreted FGF4, by itself and in the absence of cells, is stable over at least 3 days and thus – according to the Stokes-Einstein equation (see STAR Methods) – can diffuse over millimeters (see STAR Methods).

Article Snippet: After 2 days of culturing in N2B27, we added 500 nM of Retinoic Acid, and one or combinations of the following recombinant proteins to the medium: 200 ng/mL of recombinant mouse FGF4 (R&D Systems, #7486-F4), 200 ng/mL of recombinant human FGF5 (R&D Systems, #237-F5), 100 ng/mL of recombinant mouse PDGFA (Novus, NBP1-43148), 100 ng/mL of recombinant mouse VEGFB 186 (Novus, #767-VE), 100 ng/mL of recombinant mouse VEGFA (Novus, #493-MV), 500 ng/mL of recombinant human CYR61/CCN1 (Novus, #4055-CR), 500 ng/mL of recombinant human CTGF/CCN2 (Novus, #9190-CC), 200 ng/mL of recombinant mouse CLU (Novus, #2747-HS), 500 ng/mL of recombinant human HSPA8/HSC70 (Novus, #NBP1-30278), 1000 ng/mL of recombinant human Cyclophilin A (PPIA) (Novus, #NBC1-18425), or 2000 ng/mL of mouse recombinant SCF (STEMCELL, #78064).

Techniques: Enzyme-linked Immunosorbent Assay, Incubation, Concentration Assay, Control

Journal: bioRxiv

Article Title: Centimeter-scale quorum sensing dictates collective survival of differentiating embryonic stem cells

doi: 10.1101/2020.12.20.423651

Figure Lengend Snippet: To further support the idea that diffusion alone spreads the cell-secreted factors in our experiments, we determined how fast a droplet of a dye molecule of a known weight spreads in a differentiation medium without any cells, under the same incubation conditions as our cell cultures. (A) We used a gel loading dye (DNA Gel Loading Dye 6X, Thermo Scientific, #R0611) which consists of two molecules: bromophenol blue (669.96 Da) and xylene cyanol (538.61 Da). For simplicity, our calculations below will assume that the dye consists of only the heavier molecule, bromophenol blue. We injected a single, 0.5-μL droplet of the dye at the center of a 6-cm diameter plate that contained 5-mL of transparent N2B27 medium, either at room temperature (20°C) or pre-warmed at 37°C. We used a wide-field microscope to make a time-lapse movie with a bird’s eye view and snapshots every 10-seconds. Three snapshots (at 0, 30 and 60 seconds; all done at 37°C) of a single droplet of the dye shows the droplet expanding. Scale bar = 200 μm. (B) We determined the diffusion constant D of the dye in two ways: using the time-lapse movie and from theory. In the plots, three different colors represent three independent experiments. To determine D from the movies, we tracked the visible droplet boundary over time in a movie to plot the droplet area over time (shown in the two plots here at 37°C and 20°C). As shown, the droplet area linearly increased over time, which is consistent with pure diffusion (pure Brownian motion) since the area of a droplet is proportional to the mean squared displacement of a particle. Specifically, for a particle that undergoes a pure three-dimensional diffusion (Brownian motion), its mean squared displacement 〈 R 2 〉 at time t is: 〈 R 2 〉 = 6 Dt . Let A be the 2-dimensionally projected area of the droplet. Then, and hence, , where A slope is the slope of the linear fits to the droplet area as shown in the two plots here. From these fits, the experimentally determined diffusion constants D exp at 37°C and 20°C are 51.5 ± 17.8 μm 2 /s and 23.5 ± 3.9 μm 2 /s respectively ( n = 3; error bars are s.e.m.). As a comparison, we determined the diffusion constant D from theory - via the Stokes-Einstein equation which states, where k is the Boltzmann constant, T is temperature, η is the medium’s dynamic viscosity, and r dye is the radius of the dye molecule. For water, η = 0.000692 kg/m·s at 37°C and η = 0.001003 kg/m·s at 20°C (from BioNumbers (74)). We conservatively estimated r dye by noting that bromophenol blue consists of ~10 carbon-carbon bonds which would mean that the dye molecule’s diameter is 10 x 0.126 nm. For simplicity, we assume that r dye = 1 nm . The Stokes-Einstein equation then states that the dye’s diffusion constants D theory at 37°C and 20°C are 328.1 μm 2 /s and 214.0 μm 2 /s respectively. Hence, D exp < D theory . The fact that our analysis relies on the visible (by eye) boundary of the expanding droplet would underestimate the D exp since the dye must be spreading at least as fast as the boundary does. More importantly, if there were significant convection currents in the liquid medium, then D exp would be much larger than the measured value. This argues against there being any significant liquid convection in our cell-culture media. In other words, the dye-based experiment strongly indicates that cell-secreted survival-promoting factors spread out by pure diffusion rather than by convection currents which, according to the dye, are negligible in our cell-culture conditions. Furthermore, note that and ; the experimental and theoretical values for pure diffusion closely match (proportional to a factor on the order of one). (C) Based on Brownian motion in 3 dimensions with the experimentally determined diffusion constant, the dye molecule has a mean squared displacement of 〈 R 2 〉 = 1 mm 2 after (with D = D exp ( dye , 37° C) = 51.5 μm 2 /s). The same calculation, but now based on the Stokes-Einstein estimate of the diffusion constant would yield t dye = 8.5 minutes for a 1 mm 2 mean squared displacement (with D theory ( dye , 37°C) = 328.1 μm 2 /s). Hence the theory predicts a faster spreading of dye than experimentally observed - again, arguing against liquid convection or any other mechanism besides diffusion helping to spread the dye. A secreted molecule of 100 kDa would have a mean squared displacement of 1 mm 2 after time, , where we estimate the radius of the molecule to be r secreted = 20 nm (see STAR Methods). Hence the two days taken to observe appreciable amount of FGF4 and other survivalpromoting factor(s) traveling millimeters and accumulating is consistent with the t secreted calculated here (i.e., if t secreted were much larger than two days, then we should not be observing the survival-factors travelling by millimeters within two days).

Article Snippet: After 2 days of culturing in N2B27, we added 500 nM of Retinoic Acid, and one or combinations of the following recombinant proteins to the medium: 200 ng/mL of recombinant mouse FGF4 (R&D Systems, #7486-F4), 200 ng/mL of recombinant human FGF5 (R&D Systems, #237-F5), 100 ng/mL of recombinant mouse PDGFA (Novus, NBP1-43148), 100 ng/mL of recombinant mouse VEGFB 186 (Novus, #767-VE), 100 ng/mL of recombinant mouse VEGFA (Novus, #493-MV), 500 ng/mL of recombinant human CYR61/CCN1 (Novus, #4055-CR), 500 ng/mL of recombinant human CTGF/CCN2 (Novus, #9190-CC), 200 ng/mL of recombinant mouse CLU (Novus, #2747-HS), 500 ng/mL of recombinant human HSPA8/HSC70 (Novus, #NBP1-30278), 1000 ng/mL of recombinant human Cyclophilin A (PPIA) (Novus, #NBC1-18425), or 2000 ng/mL of mouse recombinant SCF (STEMCELL, #78064).

Techniques: Diffusion-based Assay, Incubation, Injection, Microscopy, Comparison, Viscosity, Convection, Cell Culture

Journal: bioRxiv

Article Title: Centimeter-scale quorum sensing dictates collective survival of differentiating embryonic stem cells

doi: 10.1101/2020.12.20.423651

Figure Lengend Snippet: Data for 46C cells differentiating towards NE lineage in N2B27+RA that were previously self-renewing in serum+LIF (see STAR Methods). With realtime quantitative PCR (primers in ), we measured anti-apoptotic, pro-apoptotic, and YAP1-mediated cell-signaling genes over the course of differentiation. Normalization of expression values: for each gene g , we first divided its expression level by the expression level of Gapdh , resulting in a value N g . For each population, we divided its N g by the low-density population’s N g on day 1 to get the final, normalized expression level μ which is plotted in here in all graphs. Thus, “1x” is the expression level of the low-density population on the first day after starting differentiation. We examined four populations: (1) high-density population (5172 cells/cm 2 ); (2) low-density population (862 cells/cm 2 ); (3) low-density population that we rescued from extinction by transplanting it, after two days, into the high-density population’s medium; and (4) low-density population that we rescued after adding 200 ng/mL recombinant mouse FGF4 to its differentiation medium on day 0. (A) Expression levels of two anti-apoptotic genes, Bcl2 (left graph) and Mdm2 (right graph). Data for Bcl2 is a replicate of the data shown in which we show here for comparison with Mdm2 . Both Bcl2 and Mdm2 show increased expressions (more anti-apoptotic) for high-density population (blue) and low-density population that was rescued by the medium of the high-density population after the 2nd day (black) or with FGF4 (green). Low-density population that goes extinct (red) shows nearly constant, low expression level of both genes. No data for 4th day is shown for the low-density population because it becomes extinct after the 3rd day (there were already barely any cells left for the 3rd day data shown here). (B) Expression levels of two pro-apoptotic genes - Bax (left graph) and Bbc3 (right graph). Color scheme is the same as in (A). The high-density population initially has a higher Bbc3 expression than the low-density population but eventually down-regulates and has lower Bbc3 expression than the low-density population. The low-density population, in turn, gradually increases its Bbc3 expression over time, up to the moment of extinction (~ day 3). Note that the rescued low-density population keeps its Bbc3 expression level low, past day 2 (which is when it receives the medium from a high-density population) and has nearly same low Bbc3 expression as the high-density population after being rescued. Note that differentiation is known to increase expression of apoptotic genes. (C) Expression levels of two cell-signaling genes that are upregulated by Yap1, Cyr61 (left graph) and Amotl2 (right graph). Data for Cyr61 is a replicate of the data shown in . Only the high-density and the rescued low-density populations gradually increase the expression levels of both genes whereas the low-density population that heads towards extinction (red) maintains a nearly constant, low expression of both genes (consistent with our findings in that secreted factors that are abundant for high-density populations increase YAP1 activity (and thus upregulate expression of Cyr61 and Amotl2 ). In all the plots, n = 3; Error bars are s.e.m.

Article Snippet: After 2 days of culturing in N2B27, we added 500 nM of Retinoic Acid, and one or combinations of the following recombinant proteins to the medium: 200 ng/mL of recombinant mouse FGF4 (R&D Systems, #7486-F4), 200 ng/mL of recombinant human FGF5 (R&D Systems, #237-F5), 100 ng/mL of recombinant mouse PDGFA (Novus, NBP1-43148), 100 ng/mL of recombinant mouse VEGFB 186 (Novus, #767-VE), 100 ng/mL of recombinant mouse VEGFA (Novus, #493-MV), 500 ng/mL of recombinant human CYR61/CCN1 (Novus, #4055-CR), 500 ng/mL of recombinant human CTGF/CCN2 (Novus, #9190-CC), 200 ng/mL of recombinant mouse CLU (Novus, #2747-HS), 500 ng/mL of recombinant human HSPA8/HSC70 (Novus, #NBP1-30278), 1000 ng/mL of recombinant human Cyclophilin A (PPIA) (Novus, #NBC1-18425), or 2000 ng/mL of mouse recombinant SCF (STEMCELL, #78064).

Techniques: Real-time Polymerase Chain Reaction, Expressing, Recombinant, Comparison, Activity Assay