Journal: Oncogene

Article Title: Targeting chemoresistant colorectal cancer via systemic administration of a BMP7 variant

doi: 10.1038/s41388-019-1047-4

Figure Lengend Snippet: BMP7v treatment promotes CR-CSC differentiation. a Phase-contrast microscopy analysis of CD44v6 + CRC sphere cells treated with BMP7v at the indicated time points. One representative of CSC#1, 2, 4, 5, 7, and 23–26 is shown. The scale bar represents 20 µm. b Percentage of CK20 positive cells in CD44v6 + CR-CSCs treated with vehicle or BMP7v up to 21 days evaluated by immunofluorescence analysis. Data are expressed as mean ± SD of experiments performed in 15 CRC sphere cell lines (CSC#1–3, 5–7, 10,11, 14–16, 18, 25, 33, and 40). c Flow cytometry analysis of CD133/CD44v6 on CRC sphere cells treated with vehicle or BMP7v for 14 days. Data reported are mean ± SD of 15 CRC sphere cell lines analyzed (CSC#1–8, 10,11, 14–16, 18, and 25). d (left panels) Immunofluorescence analysis of CDX2 on CR-CSCs upon 14 days of BMP7v treatment. One representative of CSC# 3, 9, and 21 is shown. Nuclei were stained with Toto-3 (blue color). The scale bars represent 20 µm. (right panel) Percentage of CDX2 positive cells in CD44v6 + CR-CSCs treated with vehicle or BMP7v up to 14 days evaluated by immunofluorescence analysis. Data are expressed as mean ± SD of experiments performed in CSC# 3, 9, and 21. e Flow cytometry analysis of TOP-dGFP or CD44v6 in enriched CD44v6 + sphere cells treated with BMP7v up to 14 days. One representative experiment of CSC#1, 2, 4, 7, and 10 is shown. f Phase-contrast microscopy analysis of TOP-dGFP CRC sphere cells grown in matrigel drops and treated with vehicle, BMP7v or FBS for 14 days. One representative of CSC# 8, 9, and 11 is shown. The scale bar represents 100 µm. g Immunofluorescence analysis of E-cadherin, vimentin, and β-catenin (green color) in CD44v6 + CRC cells exposed to vehicle or BMP7v for 14 days. One representative experiment performed in cells as in e is shown. Nuclei were stained with Toto-3 (blue color). The scale bars represent 20 µm. h Migrating CD44v6 + and CD44v6 − cells treated with vehicle or BMP7v up to 48 h. Data are shown as mean ± SD of three independent experiments performed in five CRC sphere cell lines (CSC#1, 5, 7, 10, and 12). i Cell viability percentage of enriched CD44v6 + and CD44v6 − cells treated with vehicle or BMP7v up to 96 h. Data are shown as mean ± SD of different experiments performed in CSC#1, 2, 4, 7, and 10. j Cell cycle analysis in CD44v6 + CR-CSCs exposed to vehicle or BMP7v for 72 h. The data show percentage of cell number in sub-G0, G0/G1, S, and G2/M phases. Data are expressed as mean ± SD of three independent experiments performed in five different CRC sphere cell lines as in e . k Immunoblot analysis of PARP, cleaved PARP (cPARP), Caspase-3 (Casp-3), cleaved Caspase-3 (cCasp-3), Bcl-2, Bcl-xL in CD44v6 + , and CD44v6 − enriched cells treated as in e for 72 h. β-actin was used as loading control. One representative experiment performed in three different CRC sphere cell lines (CSC#1, 4, and 7)

Article Snippet: Following blocking with 3% bovine serum albumin (BSA) for 30 min, cells were exposed overnight at 4 °C to BMP7 (MAB3541, mouse, IgG2 b , R&D system), LGR5 (GPR49, rabbit, IgG, Abgent), CDX2 (MAB3665, mouse, IgG1, R&D Systems), CK20 (NCL-L-CK20, mouse, IgG2 k , Novocastra Leica), E-cadherin (#3195, rabbit, IgG, CST), vimentin (#5741, rabbit, IgG, CST), β-catenin (MAB1329, mouse, IgG2 b , R&D Systems), CD44v6 (BBA13, clone 2F10, mouse, IgG1, R&D system), BMPR1A (MAB2406, mouse, IgG2 b , R&D Systems), BMPR1B (MAB505, mouse, IgG2 a , R&D Systems) and, BMPR2 (MAB811, mouse, IgG2 b , R&D Systems) antibodies or isotype-matched controls (IMCs).

Techniques: Microscopy, Immunofluorescence, Flow Cytometry, Staining, Cell Cycle Assay, Western Blot, Control

Journal: PLoS ONE

Article Title: Intestinal Stem Cell Markers in the Intestinal Metaplasia of Stomach and Barrett’s Esophagus

doi: 10.1371/journal.pone.0127300

Figure Lengend Snippet: CDX2 and ISC marker expression in 28 fresh frozen-non-tumorous gastric tissues by quantitative real-time PCR (qRT-PCR) analysis. (A) Gastric mucosae show a wide range of CDX2 expression levels, representing the various degrees of intestinal metaplasia. OLFM4 ( p < 0.0001, r 2 = 0.56) (B) and EPHB2 ( p < 0.0001, r 2 = 0.52) (C) expression increases significantly along with CDX2 levels, whereas BMI1 ( p = 0.0002, r 2 = 0.42) (D) decreases with CDX2 expression.

Article Snippet: CDX2 cDNA (pCMV6-CDX2) was purchased from OriGene (Rockville, MD, USA).

Techniques: Marker, Expressing, Real-time Polymerase Chain Reaction, Quantitative RT-PCR

Journal: PLoS ONE

Article Title: Intestinal Stem Cell Markers in the Intestinal Metaplasia of Stomach and Barrett’s Esophagus

doi: 10.1371/journal.pone.0127300

Figure Lengend Snippet: ISC expression in histologically distinct gastric lesions (n = 20), including normal antrum without intestinal metaplasia (IM) (n = 4), chronic active gastritis without IM (n = 3), gastro-intestinal mixed (GI) type IM (n = 6), solely intestinal (I) type IM (n = 5), and small intestine (n = 2). Hematoxylin and eosin staining of each gastric lesion (Magnification ×100, A). Claudin 18 expression gradually decreases ( p < 0.0001, B), while CDX2 ( p < 0.0001, C) increases with intestinal metaplastic progression. Among ISC markers, OLFM4 ( p = 0.008, D) and EPHB2 ( p = 0.001, E) expression gradually increases with metaplasia while BMI1 ( p = 0.002) (F) and LRIG1 ( p = 0.0006, G) expression is reduced.

Article Snippet: CDX2 cDNA (pCMV6-CDX2) was purchased from OriGene (Rockville, MD, USA).

Techniques: Expressing, Staining

Journal: PLoS ONE

Article Title: Intestinal Stem Cell Markers in the Intestinal Metaplasia of Stomach and Barrett’s Esophagus

doi: 10.1371/journal.pone.0127300

Figure Lengend Snippet: Both BE and adenocarcinoma sections from patients with adenocarcinoma of gastroesophageal junction (A) (n = 2) express higher levels of CDX2 (***, p < 0.005) (B) and intestinal stem cell (ISC) markers including OLFM4 (**, p < 0.01) (C), EPHB2 (*, p < 0.05) (D), and PROM1 (*, p < 0.05) (E) compared to normal esophageal mucosa. RNA in situ hybridization demonstrates that LGR5 expression in basal metaplastic glands (G) colocalizes with ASCL2 (H) and OLFM4 (I). (A) and (F) show representative sections stained with hematoxylin and eosin. Arrowheads refer to intestinal-like stem cells that express all three markers. Magnification: A, ×1.25; F ×40; G-I ×400. Eso, esophagus; BE, Barrett’s esophagus; EC, esophageal cancer.

Article Snippet: CDX2 cDNA (pCMV6-CDX2) was purchased from OriGene (Rockville, MD, USA).

Techniques: RNA In Situ Hybridization, Expressing, Staining

Journal: Communications Biology

Article Title: PHD2 safeguards modest mesendoderm development

doi: 10.1038/s42003-024-06824-z

Figure Lengend Snippet: a The schematic diagram showing the overall design of embryoid formation with different mESC lines. b The immunostaining against T on the embryoids formed with the WT ( n = 4), PHD2-KO ( n = 4), and PHD2-OE mESCs ( n = 4), respectively. c The schematic diagram showing the overall design of embryoid formation with a mixture of the WT and PHD2-KO mESCs that were labeled with GFP and mCherry, respectively. d The immunostaining against GFP and mCherry on the embryoids ( n = 6); The dashed line represents the exclusion of extraembryonic endoderm tissue from the whole embryo. Scale bars, 100 μm; BF bright field, A anterior, P posterior, WT wild type, KO PHD2-KO, OE PHD2-OE, iCdx2 inducible CDX2 expression, iGata4 inducible GATA4 expression.

Article Snippet: Phd2 , Gata4 , and Cdx2 genes were synthesized by the GenScript Biotech Corporation, with the Phd2 gene sequence codon-optimized.

Techniques: Immunostaining, Labeling, Expressing