Journal: American Journal of Clinical and Experimental Urology

Article Title: Patient derived models of bladder cancer enrich the signal of the tumor cell transcriptome facilitating the analysis of the tumor cell compartment

doi:

Figure Lengend Snippet: Spheroid and organoid media components

Article Snippet: Base Media Invitrogen 12637-010 - Advanced DMEM Invitrogen 11320-033 DMEM/F12 - B27 Invitrogen 17504-044 2× diluted 50× diluted N -acetylcysteine Sigma A9165 - 1.25 mM EGF Peprotech AF-100-15 10 ng/ml 5 ng/ml Noggin Peprotech 120-10C - 100 ng/ml R-spondin 1 R&D Systems 4645-RS-025 - 500 ng/ml or 10% conditioned medium A83-01 Tocris Bioscience 2939 - 500 nM FGF10 Peprotech 100-26 - 10 ng/ml FGF2 Peprotech 100-18B 5 ng/ml 5 ng/ml Prostaglandin E2 Tocris Bioscience 2296 - 1 μM Nicotinamide Sigma N0636 - 10 mM SB202190 Sigma S7076 - 10 μM Insulin Invitrogen 12585-014 - 4 mg/ml Y-27632 dihydrochloridea Selleck Chemical S1049 - 10 μM Open in a separate window Spheroid and organoid media components For organoid growth, viable suspended cells were cultured in 5% Matrigel in defined organoid media using the base R-spondin organoid culture system [ 15 ] with growth factor conditions adapted for use with urothelial TCC ( ).

Techniques:

Journal: Journal of Biological Chemistry

Article Title: R-spondin-1 Is a Novel β-Cell Growth Factor and Insulin Secretagogue

doi: 10.1074/jbc.m110.129874

Figure Lengend Snippet: FIGURE 1. Rspo1 and cWnt signaling molecules are expressed in murine -cells. A, simplified schematic of cWnt and Rspo1 signaling showing cWnt ligand and Rspo1 binding to the Frz receptor and LRP5/6 co-receptor, as well as the intracellular protein Dishevelled and the -catenin degradation complex consisting of APC, Axin, and GSK3. B, RT-PCR analysis of Rspo1–4 mRNA in murine pancreas, islets, and MIN6 and TC -cell lines. A 100–1000-bp ladder was used. No RNA was used in the negative (-ve) control. C, relative qRT-PCR quantification of Rspo1 mRNA transcripts in murine islets, and MIN6 and TC -cells. Primerset1,exons2–3andprimerset2,exons3–4.RelativeexpressionlevelsofRspo1werenormalizedto18SrRNAexpression.(n5–30).D,RT-PCRformRNA transcripts of various cWnt signaling molecules in MIN6 -cells. A 100–1000-bp ladder was used.

Article Snippet: Proteins of interest were detected with primary antibodies targeted against mouse Rspo1 (goat IgG, 1:1000, R&D Systems), cleaved caspase3 (rabbit IgG, 1:1000, New England Biolabs), or pan-actin (rabbit IgG, 1:1000, Sigma-Aldrich).

Techniques: Binding Assay, Reverse Transcription Polymerase Chain Reaction, Control, Quantitative RT-PCR

Journal: Journal of Biological Chemistry

Article Title: R-spondin-1 Is a Novel β-Cell Growth Factor and Insulin Secretagogue

doi: 10.1074/jbc.m110.129874

Figure Lengend Snippet: FIGURE 2. Rspo1 activates cWnt signaling and increases insulin mRNA levels in MIN6 -cells. A, ratio of nuclear -catenin to nuclear PARP in MIN6 cells treated with EX4, Wnt3a, and increasing doses of Rspo1 for 30 min. A representative blot is shown. All values are expressed as fold-relative to the control (medium alone, n 4–6). B–D, relative expression analysis of c-myc (B), cyclin D1 (C), and insulin (D) mRNA levels by qRT-PCR in MIN6 -cells treated with medium alone (control), Wnt3a, or increasing doses of Rspo1 for 12 h. Data were normalized to the housekeeping gene 18 S rRNA (n 9–11) and are displayed relative to vehicle-treated controls. *, p 0.05 and **, p 0.01.

Article Snippet: Proteins of interest were detected with primary antibodies targeted against mouse Rspo1 (goat IgG, 1:1000, R&D Systems), cleaved caspase3 (rabbit IgG, 1:1000, New England Biolabs), or pan-actin (rabbit IgG, 1:1000, Sigma-Aldrich).

Techniques: Control, Expressing, Quantitative RT-PCR

Journal: Journal of Biological Chemistry

Article Title: R-spondin-1 Is a Novel β-Cell Growth Factor and Insulin Secretagogue

doi: 10.1074/jbc.m110.129874

Figure Lengend Snippet: FIGURE 3. Rspo1 stimulates -cell proliferation. A, MIN6 -cells were treated with either medium alone (control), EX4, Wnt3a or increasing doses of Rspo1 overnight, and their proliferation index was determined by [3H]thymi- dine incorporation assay (n 14–33). B, dispersed murine islet cells were treated with medium alone (control), EX4 or Rspo1for 48 h, and BrdU was added for the last 24 h. Cells were then fixed and co-stained for insulin and BrdU. Proliferative index was determined as the number of BrdU- and insulin- positive cells over total insulin-positive cells, and data are presented as fold of control (n 4). *, p 0.05; **, p 0.01.

Article Snippet: Proteins of interest were detected with primary antibodies targeted against mouse Rspo1 (goat IgG, 1:1000, R&D Systems), cleaved caspase3 (rabbit IgG, 1:1000, New England Biolabs), or pan-actin (rabbit IgG, 1:1000, Sigma-Aldrich).

Techniques: Control, Staining

Journal: Journal of Biological Chemistry

Article Title: R-spondin-1 Is a Novel β-Cell Growth Factor and Insulin Secretagogue

doi: 10.1074/jbc.m110.129874

Figure Lengend Snippet: FIGURE 4. Rspo1 inhibits cytokine-induced -cell apoptosis. A and B, effects of Rspo1 on activated, cleaved caspase-3 in MIN6 -cells (A) or TUNEL in dispersed murine -cells (B). Cells were incubated in serum-free medium overnight, pretreated with medium alone (control), EX4, Wnt3a, or the spec- ified doses of Rspo1 for 18 h, and then incubated without (basal) or with a combined cytokine mixture for a further 18 h. MIN6 -cells were analyzed by immunoblotting for cleaved caspase-3 and pan-actin (n 4–8). A represen- tative blot is shown. Dispersed islet cells were fixed, and then co-stained for insulin and TUNEL (n 6). Apoptotic index was expressed as fold change relative to the basal control group. *, p 0.05 and **, p 0.01 when com- pared with control (basal); #, p 0.05 and ##, p 0.01 when compared with control cytokines.

Article Snippet: Proteins of interest were detected with primary antibodies targeted against mouse Rspo1 (goat IgG, 1:1000, R&D Systems), cleaved caspase3 (rabbit IgG, 1:1000, New England Biolabs), or pan-actin (rabbit IgG, 1:1000, Sigma-Aldrich).

Techniques: TUNEL Assay, Incubation, Control, Western Blot, Staining

Journal: Journal of Biological Chemistry

Article Title: R-spondin-1 Is a Novel β-Cell Growth Factor and Insulin Secretagogue

doi: 10.1074/jbc.m110.129874

Figure Lengend Snippet: FIGURE 5. Rspo1 stimulates insulin secretion in MIN6 -cells and isolated mouse islets. A, insulin secretory response to Rspo1 in MIN6 -cells (n 5 - 12) was tested by static incubation of medium containing medium alone (control), EX4, Wnt3a, or indicated doses of Rspo1 for 2 h with high glucose. Insulin in the medium was measured by radioimmunoassay, and the results were normalized to total protein content. (inset: MIN6 -cells were treated with or without Rspo1 (34.5 nM) under low (2 mM) or high glucose (25 mM) conditions (n 6–12). Data were normalized to total protein content and expressed as fold of low glucose alone). B, insulin secretion in isolated mouse islets was determined after 2 h incubation with low or high glucose and with or without Rspo1. The results were normalized to total protein content and expressed as fold of low glucose alone. *, p 0.05; **, p 0.01; ***, p 0.001 compared with control values, @@@, p 0.001 for 34.5 nM compared with 3.45 nM Rspo1, and #, p 0.05 and ###, p 0.001 for high glucose compared with high glucose in presence of 34.5 nM Rspo1.

Article Snippet: Proteins of interest were detected with primary antibodies targeted against mouse Rspo1 (goat IgG, 1:1000, R&D Systems), cleaved caspase3 (rabbit IgG, 1:1000, New England Biolabs), or pan-actin (rabbit IgG, 1:1000, Sigma-Aldrich).

Techniques: Isolation, Incubation, Control, RIA Assay

Journal: Journal of Biological Chemistry

Article Title: R-spondin-1 Is a Novel β-Cell Growth Factor and Insulin Secretagogue

doi: 10.1074/jbc.m110.129874

Figure Lengend Snippet: FIGURE 6. Rspo1 is regulated by EX4 in the -cell. A, MIN6 -cells were treated with EX4 at indicated con- centrations under low or high glucose conditions for 8 h. mRNA levels of Rspo1 were examined by relative qRT-PCR using 18 S as the internal control and then normalized to control (5 mM glucose without EX4 at the 0 h time point). B, MIN6 -cells were incubated in high glucose conditions with medium alone (control) or EX4 for the indicated times. C, protein levels of Rspo1 and actin were determined by immunoblot of MIN6 -cells treated with medium alone (control) or EX4 for 8 or 12 h. Optical densities of Rspo1 were normalized to that of pan-actinandwerefurthernormalizedtotheirappropriatecontrols.Arepresentativeblotisshownforthe12-h time point. D, qRT-PCR for Rspo1 mRNA expression in TC -cells after incubation with medium alone (control) orEX4fortheindicatedtimes.Relativeexpressionvalueswerenormalized18SrRNAandthentothe4hcontrol group. E, qRT-PCR for Rspo1 mRNA levels in mouse islets after incubation with medium alone (control) or EX4 for 4 h. Relative expression values were normalized to the control group. F, MIN6 -cells were treated with or without EX4 and with various inhibitors, as indicated, for 8 h. Relative expression values for Rspo1 were nor- malized to 18 S rRNA and then to the control treatment. *, p 0.05; **, p 0.01.

Article Snippet: Proteins of interest were detected with primary antibodies targeted against mouse Rspo1 (goat IgG, 1:1000, R&D Systems), cleaved caspase3 (rabbit IgG, 1:1000, New England Biolabs), or pan-actin (rabbit IgG, 1:1000, Sigma-Aldrich).

Techniques: Quantitative RT-PCR, Control, Incubation, Western Blot, Expressing