Journal: STAR Protocols

Article Title: Functional assays with human patient-derived enteroid monolayers to assess the human gut barrier

doi: 10.1016/j.xpro.2021.100680

Figure Lengend Snippet: Preparation of Human monolayer EDMs media (100 mL) (stored at 4°C for 2–4 days)

Article Snippet: Embed the living stem cells into a basement membrane matrix (Matrigel) dome and cultured using 50% stem cell-enriched WRN conditioned media (WNT 3a, R-spondin, and Noggin) (prepared from L-WRN cells, ATCC CRL-3276 TM ) containing specific components such as Y27632 (ROCK inhibitor) and SB431542 (an inhibitor for TGF-β type I receptor), nicotinamide, A83-01 (an ACTIVIN /NODAL/TGF-β pathway inhibitor), Epidermal growth factor, and SB202190 (a p38 MAPK inhibitor) to support the growth and long-term cultures of 3D organoids ( , , , ).

Techniques: Concentration Assay

Journal: Translational Oncology

Article Title: IDKK1 inhibits canonical Wnt signaling in human papillomavirus-positive penile cancer cells

doi: 10.1016/j.tranon.2021.101267

Figure Lengend Snippet: Wnt signaling in PeCa cell lines: Wnt signaling was analyzed using TOP-flash Assays in C33a (A), P2 (B), L2 (C), L3 (D) cells. Cells were transfected with M50 TOP plasmid or M51 FOP plasmid. Cells were treated with 30 mM LiCl, 50% (v/v) WNT3a-conditioned media or WNT3a- and (10% (v/v)) RSPO1-conditioned media 24 h after. Next day, Wnt signaling activity was measured by luciferase assay and results normalized to the total protein amount measured by Bradford assay. localization of β-catenin after Wnt signaling stimulation was analyzed in monolayer cultured PeCa cell lines using indirect immunofluorescence (E) and IHC on FFPE slides of organotypic 3D cultures (F). The 3D cultures were stimulated 24 h prior to 4% PFA fixation. Results are displayed of three independent experiments run in triplicates. Significant differences were calculated by one-way Anova and Tukey's multiple test and illustrated by asterisks (* p < 0.05; ** p < 0.01; **** p < 0.0001).

Article Snippet: Cell lines for producing Wnt3a (L-Wnt3A (ATCC®CRL-2647TM)- and RSPO1 (Cultrex® HA-R, Spondin 1-Fc 293T Cells)-conditioned media were obtained in 2018 by ATCC (Manassas, USA) and R&D (Minneapolis, USA), respectively.

Techniques: Transfection, Plasmid Preparation, Activity Assay, Luciferase, Bradford Assay, Cell Culture, Immunofluorescence

Journal: Translational Oncology

Article Title: IDKK1 inhibits canonical Wnt signaling in human papillomavirus-positive penile cancer cells

doi: 10.1016/j.tranon.2021.101267

Figure Lengend Snippet: Expression of functional DKK1 in PeCa cell lines: The expression of DKK genes was analyzed using qRT-PCR (A). Protein expression of DKK1 was analyzed by indirect immunofluorescence (B) on monolayer-cultured PeCa cell lines, ELISA (C) of supernatants and IHC (D) on FFPE-slides of organotypic 3D cultures. Nuclei were counterstained with hematoxylin (D) and DAPI (B). The staining was documented in 20x magnification. (E) Impact of PeCa cell line-conditioned media on Wnt signaling in C33a cells was analyzed. C33a cells were transfected with M50 TOP plasmid and pEYFP-C1 plasmid using CaCl 2 . Next day, cells were treated with conditioned media and stimuli. Luciferase activity was measured 24 h later. Impact of PeCa-conditioned media on Wnt signaling during Wnt3a stimulation (F) and WNT3a+RSPO1 stimulation (G). H) TOP-flash Assays using a DKK1 targeting antibody (DKK1-mAb, 0.75 μg/ml) to neutralize DKK1 in PeCa-cell line conditioned media. Impact of DKK1-mAb on Wnt signaling during Wnt3a (I) and WNT3a + RSPO1 stimulation (J). Data in A were generated of three independent experiments of three independent cDNA/cell line. Results are shown as mean +/- SEM of copies/1 × 10 6 copies RPL13a (A), concentration (ng/ml) in (C) and relative luciferase activity (normalized to medium control) in (E-J) of three independent experiments, representative pictures of three independent experiments run in triplicates (B) and of three independent cultures run in duplicates (D). Color-coding indicates treatment in E-G and cell lines in H-J. Significant differences were calculated by one-way Anova and Tukey's multiple test and illustrated with asterisks (* p < 0.05; ** p < 0.01; *** p < 0.001: **** p < 0.0001). NFK = normal foreskin keratinocytes, n.d. = not detected, sup = supernatant.

Article Snippet: Cell lines for producing Wnt3a (L-Wnt3A (ATCC®CRL-2647TM)- and RSPO1 (Cultrex® HA-R, Spondin 1-Fc 293T Cells)-conditioned media were obtained in 2018 by ATCC (Manassas, USA) and R&D (Minneapolis, USA), respectively.

Techniques: Expressing, Functional Assay, Quantitative RT-PCR, Immunofluorescence, Cell Culture, Enzyme-linked Immunosorbent Assay, Staining, Transfection, Plasmid Preparation, Luciferase, Activity Assay, Generated, Concentration Assay, Control