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: 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: Neurobiology of Pain

Article Title: circRNA landscape in dorsal root ganglia from mice with collagen antibody-induced arthritis

doi: 10.1016/j.ynpai.2023.100142

Figure Lengend Snippet: Mechanical hypersensitivity and expression of selected circRNA targets from the microarray in the CFA model. (A) Withdrawal thresholds assessed by Von Frey filaments in mice with CFA-induced monoarthritis (n = 10). Two-way ANOVA was performed followed by Šídák's multiple comparisons test. Data are presented as mean +/-SEM, n = 10 mice/group. (B-G) Expression of the selected genes measured with qPCR in DRG collected 3 days after injection of CFA (n = 16, the missing data points indicate that the gene was not detected in the sample). Unpaired t test was preformed, and error bars represent SEM.

Article Snippet: CFA-induced monoarthritis was induced by an intra-articular (i.a.) injection of 5 μl CFA (10 mg/ml, Chondrex) into one ankle joint using a 29-G needle to mice anesthetized with isoflurane (induction, 5%; maintenance, 2.5%).

Techniques: Expressing, Microarray, Injection

Journal: Oncogene

Article Title: Transcriptome profiling of a TGF-beta-induced epithelial-to-mesenchymal transition reveals extracellular clusterin as a target for therapeutic antibodies.

doi: 10.1038/onc.2009.399

Figure Lengend Snippet: Figure 2 Validation of clusterin upregulation by semi-quantitative reverse transcription–PCR, western blot analysis and immunofluorescence microscopy. (a) Semi-quantitative reverse transcription–PCR analysis (bottom panels) of the clusterin transcript (CLU) in BRI-JM01 cells induced after 2, 4, 6 and 24 h of TGF-b1 (100 pM) exposure confirming the clusterin transcript modulation observed in the microarray experiments (The graph underneath represents normalized data obtained from the microarray experiments. Bars represent the t-test P-value of 4–6 experiments performed for each time point.) The Eef1a-1 transcript (top panel), which does not change upon TGF-b1 treatment, was used as loading control. (b) Western blot analysis of whole-cell lysates (WCL) from BRI-JM01 cells grown in the absence or presence of 100 pM TGF-b1 (24 h) confirming the upregulation of clusterin and showing the uncleaved precursor form (pCLU) of clusterin in the WCL, and the the mature processed form (sCLU) in both the WCL and the CM. Membranes containing the WCL samples were reprobed for b-actin to confirm equal loading. (c) Immunofluorescence microscopy (magnification 1000) of BRI-JM01 cells treated with 100 pM TGF-b1 (24 h) show clusterin to be close to the cell’s outer membrane (top panels) and (d) colocalized with the Golgi marker b-COP (bottom) (red, clusterin; green, b-COP; blue, diamidino phenylindole (DAPI)-stained nuclei; magnification 400). (e) Western blot of the CM confirming the increased levels of secreted clusterin (sCLU) in the medium of BRI-JM01 cells treated with 100 pM TGF-b1 (24 h). CM, conditioned medium; sCLU, secreted clusterin; TGF-b, transforming growth factor-b.

Article Snippet: Antibodies against the following proteins were purchased: clusterin (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), E-cadherin (Sigma, Minneapolis, MN, USA; R&D Systems, Oakville, Canada), ZO-1 (anti-ZO1; Chemicon, Billerica, MA, USA; Zymed, San Francisco, CA, USA), b-COP (anti-b-COP; Cedarlane, Burlington, Canada) smad2 and phospho-smad2 (both Cell Signaling Technology, Danvers, MA, USA) and b-actin (Sigma).

Techniques: Biomarker Discovery, Reverse Transcription, Western Blot, Microscopy, Microarray, Control, Membrane, Marker, Staining

Journal: Oncogene

Article Title: Transcriptome profiling of a TGF-beta-induced epithelial-to-mesenchymal transition reveals extracellular clusterin as a target for therapeutic antibodies.

doi: 10.1038/onc.2009.399

Figure Lengend Snippet: Figure 3 Antibodies against sCLU block the TGF-b1-induced loss of junctional ZO-1 and E-cadherin in BRI-JM01 cells. Immunofluorescence microscopy of ZO-1 in BRI-JM01 cells grown (a) in the absence or presence of 100 pM TGF-b1 with or without antibodies against TGF-b (anti-TGF-b, 10 nM) or clusterin (anti-clu, 8 mg/ml), or (b) in CM from untreated BRI-JM01 cells (CM-CTL) or from cells treated with 100 pM TGF-b1 for 24 h (CM-TGF-b1) both in the absence or presence of antibodies against (anti-TGF-b, 10 nM) or clusterin (anti-clu, 8 mg/ml). For all the panels: red, ZO-1; blue, diamidino phenylindole (DAPI)-stained nuclei (magnification: 400). (c) Flow cytometric evaluation of the cell-surface levels of E-cadherin expressed by BRI-JM01 cells exposed to TGF-b1 (100 pM) in the absence or presence of clusterin polyclonal IgG (anti-clu, 8 mg/ml). Cell populations expressing high ( þ ) and low () levels of cell-surface E-cadherin are indicated. CM, conditioned medium; sCLU, secreted clusterin; TGF-b, transforming growth factor-b; ZO, zona occludens.

Article Snippet: Antibodies against the following proteins were purchased: clusterin (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), E-cadherin (Sigma, Minneapolis, MN, USA; R&D Systems, Oakville, Canada), ZO-1 (anti-ZO1; Chemicon, Billerica, MA, USA; Zymed, San Francisco, CA, USA), b-COP (anti-b-COP; Cedarlane, Burlington, Canada) smad2 and phospho-smad2 (both Cell Signaling Technology, Danvers, MA, USA) and b-actin (Sigma).

Techniques: Blocking Assay, Microscopy, Staining, Expressing

Journal: Oncogene

Article Title: Transcriptome profiling of a TGF-beta-induced epithelial-to-mesenchymal transition reveals extracellular clusterin as a target for therapeutic antibodies.

doi: 10.1038/onc.2009.399

Figure Lengend Snippet: Figure 4 Purified clusterin promotes a spindle-shaped morphol- ogy and loss of junctional ZO-1 in BRI-JM01 cells. (a) Coomassie blue-stained gel under non-reducing (DTT (dithiothreitol)) and reducing ( þ DTT) conditions of purified recombinant human clusterin (b) expressed and secreted by HEK-293-E6 cells. (b) BRI- JM01 cells treated with 200 nM purified clusterin (24 h) show a spindle-shaped morphology (top; magnification 40) and loss of tight-junctional ZO-1 (red, bottom; magnification 400). HEK, human embryonic kidney; ZO, zona occludens.

Article Snippet: Antibodies against the following proteins were purchased: clusterin (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), E-cadherin (Sigma, Minneapolis, MN, USA; R&D Systems, Oakville, Canada), ZO-1 (anti-ZO1; Chemicon, Billerica, MA, USA; Zymed, San Francisco, CA, USA), b-COP (anti-b-COP; Cedarlane, Burlington, Canada) smad2 and phospho-smad2 (both Cell Signaling Technology, Danvers, MA, USA) and b-actin (Sigma).

Techniques: Staining, Recombinant

Journal: Oncogene

Article Title: Transcriptome profiling of a TGF-beta-induced epithelial-to-mesenchymal transition reveals extracellular clusterin as a target for therapeutic antibodies.

doi: 10.1038/onc.2009.399

Figure Lengend Snippet: Figure 6 Clusterin polyclonal IgG inhibits the invasive behavior of cell lines other than the BRI-JM01 cell line. Clusterin polyclonal IgG (anti-clu, 8 mg/ml) inhibits (a) the stellate morphology of 4T1 and PC3 tumor cells when cultured in Matrigel for 3 weeks (magnification 40). (b) Western blot analysis confirming the presence of sCLU in the CM of 4T1, NMuMG and PC3 cells. (c) Clusterin polyclonal IgG (anti-clu, 8 mg/ml) inhibits the Matrigel invasion of 4T1, NMuMG and PC3 cell lines ing a Transwell invasion assay. Results (expressed relative to non-treated cells) are shown as the average (±s.d.) of two independent experiments. (d) Clusterin polyclonal IgG (anti-clu, 8 mg/ml) does not significantly affect the growth-inhibitory response induced by TGF-b1 in 4T1, NMuMG and PC3 cells. Results are expressed relative to non-treated cells and are shown as the average (±s.d.) of two independent experiments performed in triplicate. (e) Western blot analysis showing the presence of sCLU in the CM of MDA-MB231LM2 cells (left panel). Clusterin polyclonal IgG (anti-clu, 8 mg/ml) inhibits the Matrigel invasion of these cells in a Transwell assay (right panel). Results (expressed relative to non-treated cells) are shown as the average (±s.d.) of two independent experiments. CM, conditioned medium; sCLU, secreted clusterin; TGF-b, transforming growth factor-b.

Article Snippet: Antibodies against the following proteins were purchased: clusterin (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), E-cadherin (Sigma, Minneapolis, MN, USA; R&D Systems, Oakville, Canada), ZO-1 (anti-ZO1; Chemicon, Billerica, MA, USA; Zymed, San Francisco, CA, USA), b-COP (anti-b-COP; Cedarlane, Burlington, Canada) smad2 and phospho-smad2 (both Cell Signaling Technology, Danvers, MA, USA) and b-actin (Sigma).

Techniques: Cell Culture, Western Blot, Transwell Invasion Assay, Transwell Assay

Journal: Oncogene

Article Title: Transcriptome profiling of a TGF-beta-induced epithelial-to-mesenchymal transition reveals extracellular clusterin as a target for therapeutic antibodies.

doi: 10.1038/onc.2009.399

Figure Lengend Snippet: Figure 7 Clusterin monoclonal antibodies inhibit the motility of 4T1 cells and significantly reduce 4T1 cell metastasis to lungs after orthotopic implantation. (a) Evaluation of the ability of in-house-generated clusterin monoclonal antibodies 11E2, 16B5, 16C11 and 20G3 at a concentration of 8 mg/ml (black bars), and a commercially available clusterin monoclonal antibody B5 and anti-peptide polyclonal IgG, C18 (white bars), to block 4T1 cell motility in a BCSM assay. The average motility (±s.d.) was determined by measuring ink clearance in 10 independent microscopic fields (per treatment) and is expressed as average ink clearance/cell/24 h relative to non-treated control cells (gray bar). The hatched line depicts the cut-off that we used to define an antibody as having neutralizing activity, which was based on the degree of inhibition caused by B5 antibody. (b) Experimental design. 4T1 cells (4 104) were injected in the left # 4 inguinal mammary gland. Animals were treated (5 mg/kg) with neutralizing (16B5, 16C11 and 11E2) clusterin antibody, non-neutralizing (20G3) clusterin antibody or saline (control) thrice a week (intraperitoneally), starting the day of cell implantation. Mice were killed on day 28. (c) The number of macroscopically grossly visible lung metastasis in the mice were quantified 28 days post tumor cell implantation and statistically analysed using the non-parametric Mann–Whitney U-test (n ¼ 10 for the saline, 20G3 and 11E2 groups; n ¼ 9 for the 16B5 and 16C11 groups). BCSM, black cellular spreading and motility.

Article Snippet: Antibodies against the following proteins were purchased: clusterin (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), E-cadherin (Sigma, Minneapolis, MN, USA; R&D Systems, Oakville, Canada), ZO-1 (anti-ZO1; Chemicon, Billerica, MA, USA; Zymed, San Francisco, CA, USA), b-COP (anti-b-COP; Cedarlane, Burlington, Canada) smad2 and phospho-smad2 (both Cell Signaling Technology, Danvers, MA, USA) and b-actin (Sigma).

Techniques: Bioprocessing, Generated, Concentration Assay, Blocking Assay, Control, Activity Assay, Inhibition, Injection, Saline, MANN-WHITNEY