Journal: bioRxiv

Article Title: Modeling motor neuron resilience in ALS using stem cells

doi: 10.1101/399659

Figure Lengend Snippet: (A) Time line depicting in vitro protocols for resistant oculomotor neurons (OMN) and vulnerable spinal motor neuron (SC MN) generation. ESC column reports the four cell lines used in this study. Patterning was performed for 5 days after ESC expansion and EB formation. mRNA-seq coupled with FACS was performed on EB dissociation day (day 9 of the protocol). Survival assay was performed after five days from EB dissociation. (B-C) SC MNs generated from E14.1 ESCs express Hb9, Islet1 and NF200, OMNs generated from E14.1 ESCs over-expressing the transcription factor Phox2a under the Nestin enhancer co-express Islet1 and NF200 in absence of Hb9. Scale bar in c 60 μm. (D) Percentage of Hb9+/Islet1+ cells in SC MN (64.1±5.2%, mean±SEM, n=4) and OMN (6.3±1.3%, mean±SEM) cultures respectively. (For quantification, experiments were run in quadruplicates including two technical replicates per experiment, with at least 120 Islet1+ cells counted per condition). (E) Microphotographs showing Islet1+/Tuj1+ cells in OMN cultures, (F) OMNs also express the specific marker Phox2a as indicated by asterisks. Scale bars in e and g 100 μm. (G) Quantification of Islet1+ over Tuj1+ cells demonstrates that half neuronal population appears to be Islet1+ (47.5±5.9%, mean±SEM, experiments performed in quadruplicates with two technical replicates each, total number of Tuj1+ cells counted: 1325). Quantification of Phox2A+ over Islet1+ cells (experiments performed in quadruplicates with two technical replicates each, total number Islet1+ cells counted: 746) indicates that 62±5.2% (mean±SEM) of the Islet1+ population is also Phox2a+. All quantifications were performed 5 days after EB dissociation (mean ± SEM). (H) PCA of OMN and SC MN samples based on all genes expressed confirmed cell differential identities. mRNA-seq analysis of OMNs and SC MNs isolated by FACS was performed after 5 days of patterning (day 9 of the protocol). (I) 1,017 differentially expressed genes were found between the two different cell types (adjusted P<0.05). Heatmap shows the top 500 most significant differentially expressed genes by adjusted p-value. (J) Heatmap of expected progenitors and motor neuron transcripts but also OMN and SC MN specific transcripts obtained in the two generated motor neuron populations. (K) Using the top 100 DEGs obtained from our RNAseq analysis we could separate datasets originating from in vivo microarray studies on early postnatal (J) and adult (L) rodent OMNs and SC MNs. (M) Venn diagram showing gene sets enriched either in brain stem cultures or OMN-specific as revealed by PAGODA analysis, (N) gene sets preferentially found in spinal cord cultures or MN-specific.

Article Snippet: The following primary antibodies were used: NF200 (1:1000, #AB5539 Millipore), Tuj1 (1:1000, MRB-435P Covance), Hb9 (1:10, #81.5C10 DSHB), Islet1 (1:500, #ab20670 abcam; 1:100, #40.2D6 DSHB), Phox2A (1:1000, gift of Prof JF Brunet), GFP (1:1000, #ab13970 abcam), phospho-AKT (Ser473) (1:50, #3787 Cell Signaling), activated beta-catenin (1:1000, #05-665 Millipore), alpha-catenin (1:1000, #AB153721 AbCam), ESYT1 (1:100, #HPA016858), BrdU (1:10, #G3G4 DSHB).

Techniques: In Vitro, Clonogenic Cell Survival Assay, Generated, Expressing, Marker, Isolation, In Vivo, Microarray

Journal: bioRxiv

Article Title: Modeling motor neuron resilience in ALS using stem cells

doi: 10.1101/399659

Figure Lengend Snippet: (A) Oculomotor neurons (OMNs) and spinal motor neurons (SC MNs) express similar mRNA levels of glutamate ionotropic receptor AMPA, NMDA and kainate type subunits. The heatmap shows log2 RPKM values of these subunits and no separate clustering of the two cell types is observed. (B-C) Immunohistochemistry performed on generated OMNs and SC MNs at D1 in vitro in control conditions; similar levels of glutamate ionotropic receptor kainate type subunit 5 (Grik5) are found in both cell types. Scale bar in d 60 μm. (D-E) Microphotographs presenting SC MN and OMN response to kainic acid induced toxicity (20 μM) for a week. Scale bars in f = 100 μm. (F) Curves represent percentages of MN survival over time in OMN and SC MN cultures. OMNs were visualized as NF200+Islet1+Hb9- clls, while SC MNs as NF200+Islet1+Hb9+ cells. OMNs show increased survival to KA toxicity at D7 (mean ± SEM, 2way ANOVA and Tukey’s multiple comparison test, F(9, 56)=2.333, *P=0.0261, SC MNs n=4; OMNs n=5) when compared to SC MNs (experiments were performed at least in quadruplicates, with technical replicates and with at least 130 motor neurons counted per condition in each experiment). Analysis of the length of neuronal processes in both oculomotor and spinal motor neuron cultures exposed to kainic acid for seven days. showed that oculomotor neurons were unaffected by kainic acid while spinal motor neurons displayed a shortening of neurites (G). (H-K) Sholl analysis was performed on OMN at D7 survival assay in control and KA20 conditions to further assess individual MN arborization complexity during toxicity. Scale bar = 100 μm. (I) Sholl mask was applied to individual OMNs after specifying the radius from the center of the soma of the neuron and created concentric circles every 25 μm of increasing radius. (J) Comparison of average number of neurite intersections of OMN in control and KA20 toxicity conditions with radial step size of 25 μm. OMNs did not show reduction in arborization (multiple t test, n = 10 per condition). (K) Schematic depicting identification of neurite segments by Sholl analysis. Color code is assigned depending on arbor localization from the soma in an inside-out manner following the given radius. Multiple intersections within the same segment display the same colour.

Article Snippet: The following primary antibodies were used: NF200 (1:1000, #AB5539 Millipore), Tuj1 (1:1000, MRB-435P Covance), Hb9 (1:10, #81.5C10 DSHB), Islet1 (1:500, #ab20670 abcam; 1:100, #40.2D6 DSHB), Phox2A (1:1000, gift of Prof JF Brunet), GFP (1:1000, #ab13970 abcam), phospho-AKT (Ser473) (1:50, #3787 Cell Signaling), activated beta-catenin (1:1000, #05-665 Millipore), alpha-catenin (1:1000, #AB153721 AbCam), ESYT1 (1:100, #HPA016858), BrdU (1:10, #G3G4 DSHB).

Techniques: Immunohistochemistry, Generated, In Vitro, Clonogenic Cell Survival Assay

Journal: Nature biotechnology

Article Title: Generation of pancreatic β cells from CD177 + anterior definitive endoderm.

doi: 10.1038/s41587-020-0492-5

Figure Lengend Snippet: Fig. 1 | Identification of CD177+ and CD275+ ADE subpopulations. a, Schematic representation of hESC differentiation toward DE. b,c, Representative FACS plots of apparently homogeneous FOXA2+/SOX17+ DE (b) showing a heterogenous population marked by CXCR4+/CD117+ cells (c) (n = 3 (b), n = 6 (c) biologically independent experiments). d–g, Gene expression profiles of CXCR4+/CD117−, CXCR4high/CD117high, CXCR4mid/CD117mid and CXCR4low/CD117low cells for FOXA2 (d), SOX17 (e), CER1 (f) and HHEX (g) (ANOVA, n = 3 biologically independent experiments). Data are represented as mean ± s.e.m.; P < 0.05 and P < 0.01. Statistically nonsignificant results are not indicated in the figure. h, Summary of the antibody screen identifying and isolating CD177 and CD275 as markers of ADE subpopulations. CXCR4 and FOXA2 are used as controls to identify the whole DE. i, hPSCs and hPSC-derived DE stained for CXCR4, CD177 and CD275 as shown by live-cell FACS (n = 10 biologically independent experiments). AA, activin A; D, day.

Article Snippet: Materials & experimental systems n/a Involved in the study Antibodies Eukaryotic cell lines Palaeontology Animals and other organisms Human research participants Clinical data Methods n/a Involved in the study ChIP-seq Flow cytometry MRI-based neuroimaging Antibodies Antibodies used Human CXCR4-PE,Miltenyi Biotech,130-098-354, dilution 1:40; Human CXCR4-APC,Miltenyi Biotech, 120-010-802, dilution 1:40; Human CD117-APC, Miltenyi Biotech, 130-091-733, dilution 1:40; Human CD117-PE, Miltenyi Biotech, 130-091-734, dilution 1:40; FOXA2-Alexa Fluor® 488, R and D, IC2400G; dilution 1:10 SOX17-APC, R and D, IC1924A; dilution 1:10 Human CD177-APC, Miltenyi Biotech, 120-017-498; dilution 1:20 Human CD275-APC, Miltenyi Biotech, 120-012-112; dilution 1:20 PE Mouse anti-PDX1, BD PharmingenTM, 562161; dilution 1:40 4 nature research | reporting sum m ary O ctober 2018 Alexa Fluor® 647 Mouse anti-Nkx6.1, BD PharmingenTM, 563338; dilution 1:40 Alexa Fluor® 647 Mouse IgG1 κ Isotype Control, BD PharmingenTM, 563023; dilution 1:40 Rabbit FOXA2, Cell signalling, 8186; dilution 1:1000 Goat SOX17 Acris/Novus GT15094, dilution 1:1000 Goat CER1 R&D Systems AF1075, dilution 1:1000 Mouse β-catenin BD 610154, dilution 1:1000 Guinea pig INSULIN Thermo Schientific PA1-26938, dilution 1:100 Guinea pig C-Peptide Abcam ab30477, dilution 1:300 Rabbit MAFA Betalogics LP9872, dilution 1:100 Rabbit MAFA ,Novus Biologicals, NB400-137, dilution 1:100 Rabbit GLUT1 Thermo Fisher PA1-37782, dilution 1:100 Goat GATA6 R&D Systems AF1700, dilution 1:1000 Mouse SOX2 Abgent / Bio Cat AM2048, dilution 1:1000 Rabbit CDX2 Santa Cruz sc-134468, dilution 1:1000 Mouse GCG Sigma G2654-.2ML, dilution 1:300 Goat PDX1 R&D Systems AF2419, dilution 1:500 Rabbit NKX6.1Novus biologicalsNBP1-49672, dilution 1:500 Goat NKX6.1R&D systemsAF5857, dilution 1:300 Rabbit p-JNK Cell signalling 4668, dilution 1:1000 Rabbit DVL2 Cell signalling 3216, dilution 1:1000 Mouse GAPDH Merck Biosciences CB1001, dilution 1:6000 Validation All primary antibodies were validated for their expression on undifferentiated cells and/or pancreatic human sections/islets.

Techniques: Gene Expression, Derivative Assay, Staining

Journal: Nature biotechnology

Article Title: Generation of pancreatic β cells from CD177 + anterior definitive endoderm.

doi: 10.1038/s41587-020-0492-5

Figure Lengend Snippet: Fig. 2 | Molecular profiling of CD177+, CD275+ and CXCR4+ DE subpopulations reveals distinct signatures. a, Summary of differentiation protocol toward DE/ADE followed by MACS to enrich for CD177, CD275 and CXCR4 populations. b, Principal component analysis showing that mRNA-derived transcriptome profiles are characteristic of different DE/ADE subpopulations (n = 3 biologically independent experiments). c–e, Bar graphs of selected and significantly enriched gene ontology terms in CD275+ versus CXCR4+ (c), CD177+ versus CD275+ (d) and CD177+ versus CXCR4+ (e) DE populations (n = 3 biologically independent experiments). Enrichment P values are calculated by HOMER findGO.pl based on the cumulative hypergeometric distribution. f,g, Validation of the microarray analysis by qPCR for noncanonical WNT/PCP components and ligands (f) and canonical WNT components and ligands (g). Data were normalized to 18S (ANOVA, n = 3 biologically independent experiments). Data are represented as mean ± s.e.m.; P < 0.05 and P < 0.01. Statistically nonsignificant results are not indicated in the figure. h,i, Western blot analysis (h) and quantification (i) of WNT/PCP components such as p-JNK and DVL2 in ADE subpopulations (n = 3 biologically independent experiments). GAPDH is used as a loading control. Data are represented as mean ± s.e.m. j, Immunofluorescence analysis validated the exclusive localization of β-catenin in the membrane in CD177+ ADE cells and in the cytoplasm and nucleus in CD275+ ADE and CXCR4+ DE cells (n = 3 biologically independent experiments). FOXA2 is used as a nuclear marker. Scale bars, 20 µm and 10 µm in inset. PC1/2, principal component 1/2.

Article Snippet: Materials & experimental systems n/a Involved in the study Antibodies Eukaryotic cell lines Palaeontology Animals and other organisms Human research participants Clinical data Methods n/a Involved in the study ChIP-seq Flow cytometry MRI-based neuroimaging Antibodies Antibodies used Human CXCR4-PE,Miltenyi Biotech,130-098-354, dilution 1:40; Human CXCR4-APC,Miltenyi Biotech, 120-010-802, dilution 1:40; Human CD117-APC, Miltenyi Biotech, 130-091-733, dilution 1:40; Human CD117-PE, Miltenyi Biotech, 130-091-734, dilution 1:40; FOXA2-Alexa Fluor® 488, R and D, IC2400G; dilution 1:10 SOX17-APC, R and D, IC1924A; dilution 1:10 Human CD177-APC, Miltenyi Biotech, 120-017-498; dilution 1:20 Human CD275-APC, Miltenyi Biotech, 120-012-112; dilution 1:20 PE Mouse anti-PDX1, BD PharmingenTM, 562161; dilution 1:40 4 nature research | reporting sum m ary O ctober 2018 Alexa Fluor® 647 Mouse anti-Nkx6.1, BD PharmingenTM, 563338; dilution 1:40 Alexa Fluor® 647 Mouse IgG1 κ Isotype Control, BD PharmingenTM, 563023; dilution 1:40 Rabbit FOXA2, Cell signalling, 8186; dilution 1:1000 Goat SOX17 Acris/Novus GT15094, dilution 1:1000 Goat CER1 R&D Systems AF1075, dilution 1:1000 Mouse β-catenin BD 610154, dilution 1:1000 Guinea pig INSULIN Thermo Schientific PA1-26938, dilution 1:100 Guinea pig C-Peptide Abcam ab30477, dilution 1:300 Rabbit MAFA Betalogics LP9872, dilution 1:100 Rabbit MAFA ,Novus Biologicals, NB400-137, dilution 1:100 Rabbit GLUT1 Thermo Fisher PA1-37782, dilution 1:100 Goat GATA6 R&D Systems AF1700, dilution 1:1000 Mouse SOX2 Abgent / Bio Cat AM2048, dilution 1:1000 Rabbit CDX2 Santa Cruz sc-134468, dilution 1:1000 Mouse GCG Sigma G2654-.2ML, dilution 1:300 Goat PDX1 R&D Systems AF2419, dilution 1:500 Rabbit NKX6.1Novus biologicalsNBP1-49672, dilution 1:500 Goat NKX6.1R&D systemsAF5857, dilution 1:300 Rabbit p-JNK Cell signalling 4668, dilution 1:1000 Rabbit DVL2 Cell signalling 3216, dilution 1:1000 Mouse GAPDH Merck Biosciences CB1001, dilution 1:6000 Validation All primary antibodies were validated for their expression on undifferentiated cells and/or pancreatic human sections/islets.

Techniques: Derivative Assay, Biomarker Discovery, Microarray, Western Blot, Control, Immunofluorescence, Membrane, Marker

Journal: Microbial Cell Factories

Article Title: Key role of lipid management in nitrogen and aroma metabolism in an evolved wine yeast strain

doi: 10.1186/s12934-016-0434-6

Figure Lengend Snippet: Ratios of final liquid concentrations of fermentative aromas produced by Affinity™ ECA5 and Lalvin EC1118 ® . These ratios were calculated from 16 fermentation experiments with different temperature, nitrogen and lipid contents. If the ratio value is higher than one, the compound is considered as overproduced by the evolved strain. PR propanol, ISO isobutanol, IA isoamyl alcohol, HE hexanol, ME methionol, PHE 2-phenylethanol, EA ethyl acetate, ISA isobutyl acetate, AA amyl acetate, IAA isoamyl acetate, PEA 2-phenylethylacetate, PA propanoic acid, BA butanoic acid, IBA isobutanoic acid, IVA isovaleric acid, MBA 2-methylbutanoic acid, VA valeric acid, HA hexanoic acid, OA octanoic acid, DA decanoic acid, EB ethyl butanoate, DS diethyl succinate, EL ethyl lactate, EV ethyl valerate, EH ethyl hexanoate, EO ethyl octanoate, ED ethyl decanoate, EDD ethyl dodecanoate

Article Snippet: We used the Agilent 8 × 15 k gene expression microarrays (Design ID 038619 with 40 EC1118-specific genes, Agilent Technologies, Santa Clara, CA, USA) according to the manufacturer’s instructions.

Techniques: Produced

Journal: Microbial Cell Factories

Article Title: Key role of lipid management in nitrogen and aroma metabolism in an evolved wine yeast strain

doi: 10.1186/s12934-016-0434-6

Figure Lengend Snippet: Timing of exhaustion of each nitrogen source (Point.AA0) expressed in terms of consumed sugar (g/L) for each fermentation condition. Lalvin EC1118 ® -SM70-2 mg/l of phytosterols ( closed orange circle ); Lalvin EC1118 ® -SM70-8 mg/l of phytosterols ( closed beige circle ); Lalvin EC1118 ® -SM330-2 mg/l of phytosterols ( closed blue circle ); Lalvin EC1118 ® -SM330-8 mg/l of phytosterols ( closed purple circle ); Affinity™ ECA5-SM70-2 mg/l of phytosterols ( closed orange triangle ); Affinity™ ECA5-SM70-8 mg/l of phytosterols ( closed beige triangle ); Affinity™ ECA5-SM330-2 mg/l of phytosterols ( closed blue triangle ); Affinity™ ECA5-SM330-8 mg/l of phytotserols ( closed purple triangle ). Gly glycine, Tyr tyrosine, Trp Tryptophan, Ala alanine, Arg arginine, Val valine, NH4 ammonium, Phe phenylalanine, Gln glutamine, Ser serine, Ile isoleucine, Met methionine, His histidine, Leu leucine, Glu glutamate, Thr threonine, Asp aspartate

Article Snippet: We used the Agilent 8 × 15 k gene expression microarrays (Design ID 038619 with 40 EC1118-specific genes, Agilent Technologies, Santa Clara, CA, USA) according to the manufacturer’s instructions.

Techniques:

Journal: Microbial Cell Factories

Article Title: Key role of lipid management in nitrogen and aroma metabolism in an evolved wine yeast strain

doi: 10.1186/s12934-016-0434-6

Figure Lengend Snippet: Total production of fermentative aromas for each fermentation condition

Article Snippet: We used the Agilent 8 × 15 k gene expression microarrays (Design ID 038619 with 40 EC1118-specific genes, Agilent Technologies, Santa Clara, CA, USA) according to the manufacturer’s instructions.

Techniques: Concentration Assay

Journal: Microbial Cell Factories

Article Title: Key role of lipid management in nitrogen and aroma metabolism in an evolved wine yeast strain

doi: 10.1186/s12934-016-0434-6

Figure Lengend Snippet: Changes in the specific rates of total production of isoamyl acetate in SM70 ( a ) or SM330 ( b ). Lalvin EC1118 ® −2 mg/L of phytosterol ( blue ); Lalvin EC1118 ® −8 mg/L of phytosterol ( light blue ); Affinity™ ECA5–2 mg/L of phytosterol ( red ); Affinity™ ECA5–8 mg/L of phytosterol ( pink )

Article Snippet: We used the Agilent 8 × 15 k gene expression microarrays (Design ID 038619 with 40 EC1118-specific genes, Agilent Technologies, Santa Clara, CA, USA) according to the manufacturer’s instructions.

Techniques:

Journal: Microbial Cell Factories

Article Title: Key role of lipid management in nitrogen and aroma metabolism in an evolved wine yeast strain

doi: 10.1186/s12934-016-0434-6

Figure Lengend Snippet: Production yields of acetate ester from its higher alcohol precursor

Article Snippet: We used the Agilent 8 × 15 k gene expression microarrays (Design ID 038619 with 40 EC1118-specific genes, Agilent Technologies, Santa Clara, CA, USA) according to the manufacturer’s instructions.

Techniques:

Journal: Microbial Cell Factories

Article Title: Key role of lipid management in nitrogen and aroma metabolism in an evolved wine yeast strain

doi: 10.1186/s12934-016-0434-6

Figure Lengend Snippet: Focus on the bioconversion of isoamyl alcohol to isoamyl acetate. a Changes in total isoamyl acetate production as a function of total isoamyl alcohol production in SM330 with 8 mg/L of phytosterols for Lalvin EC1118 ® ( blue ) and Affinity™ ECA5 ( red ). The arrows indicate the timing of sampling for transcriptomic analyses. b Changes in total isoamyl acetate production as a function of total isoamyl alcohol production subsequently to phytosterol additions for Lalvin EC1118 ® ( blue ) and Affinity™ ECA5 ( purple and red ). For Lalvin EC1118 ® , despite phytosterol additions, bioconversion yield between the two compounds remains identical: 0.0241 (R 2 = 0.947). For Affinity™ ECA5, four linear phases are identified; their yields of conversion are 0.0689 (R 2 = 0.990); 0.0367 (R 2 = 0.966); 0.0696 (R 2 = 0.991); and 0.0351 (R 2 = 0.955)

Article Snippet: We used the Agilent 8 × 15 k gene expression microarrays (Design ID 038619 with 40 EC1118-specific genes, Agilent Technologies, Santa Clara, CA, USA) according to the manufacturer’s instructions.

Techniques: Sampling

Journal: Nature Communications

Article Title: Hypoxia induced responses are reflected in the stromal proteome of breast cancer

doi: 10.1038/s41467-023-39287-7

Figure Lengend Snippet: Multivariate survival analysis (proportional hazards regression model) of breast cancer patients (METABRIC-Discovery cohort)

Article Snippet: Of relevance for secretome studies, we found that 40 of the PAM50 signature genes/proteins have been reported in serum or plasma (plasma proteome database; PPD: http://www.plasmaproteomedatabase.org/ ) , and/or the Human Protein Atlas—blood protein (Human Protein Atlas proteinatlas.org) .

Techniques:

Journal: Cancer immunology research

Article Title: Autocrine TGFβ is a Survival Factor for Monocytes and Drives Immunosuppressive Lineage Commitment

doi: 10.1158/2326-6066.CIR-18-0310

Figure Lengend Snippet: A. cBioportal for cancer genomics (15,16) was used to assess the correlation between TGFB1 gene expression and myeloid cell marker CD11b (ITGAM) in TCGA NSCLC (N=586) and Pearson and Spearman correlations were calculated. B. cBioportal was used to test correlations between expression of TGFB1 and myeloid cell markers CD14, CD33, CSF1R, CD11c (ITGAX) and CD103, NK cell marker CD56 (NCAM1), and neutrophil/granulocyte marker CD66b (CEACAM8) using TCGA NSCLC adenocarcinoma specimens (N=586) (ns= not statistically significant correlation). C. Examples of NSCLC tissue microarray stained for active TGFβ (green) and CD11b (red). DAPI (blue) was used to stain the nuclei. D. Correlation between CD11b and TGFβ activity immunostaining in NSCLC tissue array (N=66 samples, p=0.02; Pearson, Spearman=0.3). E. Kaplan Meyer survival curves of TCGA NSCLC patients stratified according to the expression of TGFB1 (z-score threshold=2; N=514 patients TGFB1hi, N=514 patients TGFB1lo). HR=3.4. Median survival=49.8 mo (TGFB1lo) vs 32.4 mo (TGFB1hi).

Article Snippet: Immunofluorescence A lung tumor tissue microarray (TMA) was obtained from US Biomax ( {"type":"entrez-nucleotide","attrs":{"text":"BC041114","term_id":"26996621","term_text":"BC041114"}} BC041114 ).

Techniques: Expressing, Marker, Microarray, Staining, Activity Assay, Immunostaining