Journal: eLife

Article Title: A human ESC-based screen identifies a role for the translated lncRNA LINC00261 in pancreatic endocrine differentiation

doi: 10.7554/eLife.58659

Figure Lengend Snippet:

Article Snippet: Peptide, recombinant protein , KGF/FGF7 , R and D Systems , Cat# 251 KG , .

Techniques: Flow Cytometry, Control, Recombinant, Cloning, Plasmid Preparation, Expressing, Transfection, TA Cloning, Purification, cDNA Synthesis, SYBR Green Assay, Enzyme-linked Immunosorbent Assay, Bicinchoninic Acid Protein Assay, Software, Membrane, Hybridization, DNA Extraction, Modification

Journal: The Journal of investigative dermatology

Article Title: Differentiation of human embryonic stem cells into clinically amenable keratinocytes in an autogenic environment.

doi: 10.1038/jid.2012.384

Figure Lengend Snippet: Figure 1. Characterization of H9-ebF. (a, b) Qualitative analysis of gene expression profile of (a) pluripotency marker and (b) fibroblast markers. b-Actin was taken as the loading control and run on a separate gel. (c) Results of ELISA show the KGF-I secretion by P 5–P 10 of H9-ebF. Error bars indicate the mean ±SEM (n ¼ 3). *Po0.05 versus all groups. (d, e) Immunofluorescence staining shows the expression of (d) K18 and fibronectin by H9-ebF passage 8 (scale bar ¼ 100 mm), (e) K14 by HaCaT cells–H9-ebF passage 8 in coculture (scale bar ¼ 50mm). b-Act, b-actin; CRL-1486, human embryonic palatal mesenchymal cell line; DAPI, 4,6-diamidino-2-phenylindole; FAD, F12 and Dulbecco medium; H, H9-ebF; hESC, human embryonic stem cell; H9-ebF, H9-hESC-derived fibroblast; K, HaCaT cells; KGF-I, keratinocyte growth factor-I; P, passage; 4PB, prolyl 4-hydroxylase; PC, phase contrast; Vim, vimentin.

Article Snippet: ELISA was performed using the DuoSet ELISA Development System (catalog # DY251, R&D systems) as per the manufacturer’s instruc- tions.

Techniques: Gene Expression, Marker, Control, Enzyme-linked Immunosorbent Assay, Staining, Expressing, Derivative Assay

Journal: bioRxiv

Article Title: Basal stem cell fate specification is mediated by SMAD signaling in the developing human lung

doi: 10.1101/461103

Figure Lengend Snippet: a ) Bud tip progenitor organoids from n=3 biological replicates from 12 week fetal lungs were maintained in serum-free progenitor maintenance medium (FGF7, CHIR99021, ATRA) for 56 days. Staining for markers of differentiated lung epithelial cell types determined that bud tip progenitor organoids did not contain any differentiated cell types (club cell marker SCGB1A1 (pink), neuroendocrine markers Chromagranin A (CHGA; pink) and synaptophysin (SYN; white), ciliated cell marker FOXJ1 (white), basal cell marker TP63 (pink), goblet cell marker mucin 5AC (MUC5AC; white), AECII marker ABCA3 (pink), AECI and hub progenitor cell marker HOPX (green), secretory lineage marker PLUNC (white)). The majority of bud tip progenitor organoid cells were SOX9+ (white). Scale bar represents 100 µm. b ) Organoids that had been infected with GFP lentivirus but not sorted and left to expand in basal cell expansion medium were collected after 56 days in culture and stained for differentiated epithelial cell markers. Unsorted organoids exhibited clear positive staining for club cell marker SCGB1A1 (pink) and multiciliated markers Acetylated Tubulin (white) and FOXJ1 (white). Neuroendocrine cells were clearly detected (CHGA, pink; SYN, white). Organoids also exhibited TP63+ cells (pink) and cells that stained positive for goblet cell marker MUC5AC (white). Scale bar represents 50 µm. c ) Organoids treated for 3 days DSA and 18 days DSI-expansion, FACS sorted for EGFR/F3 and clonally expanded in DSI expansion medium were stained for differentiated cell markers. Staining for AECI and hub progenitor marker HOPX (green) was negative, as was staining for AECII marker ABCA3 (pink). Organoids grew clonally, with organoids being either entirely GFP negative or GFP positive (GFP, green, second panel). Many cells exhibited positive staining for club cell marker SCGB1A1 (pink), but staining for secretory lineage marker PLUNC (white) was undetected. In EGFR+/F3+ clonal organoids, no neuroendocrine cells were detected (CHGA, pink; SYN, white), though these cells were clearly detected in unsorted organoids. Scale bar represents 50 µm. d ) Unsorted organoids also did not show any positive staining for AECI/hub progenitor marker HOPX (green), or AECII marker ABCA3 (pink). Consistent with results from sorted organoids, the secretory lineage marker PLUNC was not detected (white). Scale bar represents 50 µm. e ) Graph of the percent of GFP+ organoids versus total number of organoids from each group. The total number of organoids counted per group is reported. 2 wells of multiple organoids were counted for each biological replicate for the EGFR+/F3+ group.

Article Snippet: To maintain bud tip progenitor organoids in a progenitor state, serum-free basal medium was further supplemented with FGF7 (10 ng/mL, Recombinant Human Fibroblast Growth Factor 7; R&D Systems cat. no. 251-KG/CF), CHIR99021 (3 μM, Stem Cell Technologies cat. no. 72054), and All Trans Retinoic Acid (ATRA; 50 nM, Stemgent cat. no. 04-0021, CAS Number 302-79-4).

Techniques: Staining, Marker, Infection

Journal:

Article Title: Induction of Keratinocyte Growth Factor 1 Expression by Lipopolysaccharide Is Regulated by CD-14 and Toll-Like Receptors 2 and 4

doi: 10.1128/IAI.70.12.6541-6548.2002

Figure Lengend Snippet: LPS induces concentration-dependent induction of total CD14 protein expression. Quiescent gingival fibroblasts were stimulated for 18 h with E. coli LPS (50 ng/ml) in α-MEM plus 1% FBS. Whole-cell protein extracts were prepared from control and LPS-treated cultures, and proteins were analyzed by Western blotting with an anti-CD14 polyclonal antibody. (A) LPS (50 ng/ml) induced significant expression of CD14, a 55-kDA protein. (B) In relation to the control (α-MEM plus 1% FBS), LPS (5 and 50 ng/ml) induced a concentration-dependent increase in CD14 total protein expression. Mean ± range; n = 2.

Article Snippet: Wells were washed, and 100 μl of biotinylated anti-human KGF-1 polyclonal antibody (BAF251; 200 ng/ml; R&D Systems Inc.) was added, the wells were incubated for 2 h, and then the wells were washed.

Techniques: Concentration Assay, Expressing, Western Blot