Journal: REPRODUCTION

Article Title: Granulosal and thecal expression of bone morphogenetic protein- and activin-binding protein mRNA transcripts during bovine follicle development and factors modulating their expression in vitro

doi: 10.1530/rep-11-0150

Figure Lengend Snippet: Figure 3 Immunohistochemical staining of bovine ovary sections showing the distribution of chordin, gremlin, noggin and follistatin immunoreactivity (brown) in the granulosa and theca layer of small antral follicles (w3 to 5 mm diameter). In eachpanel the left-hand image shows an antibody-treated section while the right-hand image shows an adjacent section treated with the appropriate non-immune serum or purified IgG. The scale bar in each right panel represents 50 mm.

Article Snippet: The following primary antibodies (and appropriately matched control serum/purified IgG for adjacent tissue section) were diluted in blocking buffer and incubated with sections overnight at 4 8C: goat anti-mouse gremlin (R&D Systems) at 5 mg/ml, goat anti-mouse chordin (R&D Systems) at 5 mg/ml, rabbit anti-human noggin (Abcam Ltd, Cambridge, UK) at 5 mg/ml and rabbit anti-human follistatin (FP22; supplied by NIDDK, Baltimore, MD, USA) at 1:500 dilution.

Techniques: Immunohistochemical staining, Staining

Journal: Cells

Article Title: CHRDL1 Regulates Stemness in Glioma Stem-like Cells

doi: 10.3390/cells11233917

Figure Lengend Snippet: CHRDL1 depletion blocks stemness in GSCs. ( A ) Western Blot of NCH644 and GS-5 GSCs after stable transduction with a non-targeting shRNA (shCtrl) or specific shRNA targeting CHRDL1 (shCHRDL1) and ( B ) Quantification of several independent experiments. ( C , E , F ) Log-fraction plots of the limited dilution model of data from ( C ) NCH644 shCtrl (black) and NCH644 shCHRDL1 (red) GSCs 7 days after seeding the cell in a dilution series from 1024 to 8 cells and analyzing the data using ELDA software . ( D ) Representative microphotographs of NCH644 and GS-5 with and without CRHDL1-depletion. Scale bar: 200 µm. ( E , F ) Data from GS-5 GSCs ( E ) 7 days and ( F ) 14 days after seeding of 2048 to 16 cells. **: p < 0.01, ***: p < 0.001 ****: p < 0.0001.

Article Snippet: The following primary antibodies and dilutions were used: CHRDL1 (#AF1808, R&D Systems, Wiesbaden-Nordenstadt, Germany) 1:2000; GAPDH (#CB1001, Calbiochem, Darmstadt, Germany) 1:20,000; OLIG2 (#AF2418, R&D Biosystems) 1:5000; SOX2 (#MAB2018, R&D Systems) 1:5000; SOX9 (#ab185966, Abcam, Cambridge, UK); SMAD1 [(#6944, Cell Signaling Technology (CST), Danvers, MA, USA)] 1:1000; SMAD5 [(#12534, CST)] 1:1000; pSMAD1/5 [(#9516, CST)] 1:1000.

Techniques: Western Blot, Transduction, shRNA, Software

Journal: Cells

Article Title: CHRDL1 Regulates Stemness in Glioma Stem-like Cells

doi: 10.3390/cells11233917

Figure Lengend Snippet: Cell cycle distribution of GSCs after CHRDL1-depletion. ( A , B ) Stacked bar graph of ( A ) NCH644 and ( B ) GS-5 after stable transduction with a non-targeting shRNA (shCtrl) or specific shRNA targeting CHRDL1 (shCHRDL1). After 24 h of seeding the cells, the cell cycle distribution was analyzed. For NCH644 GSCs a significant increase in cells in ( A ′) G1-phase with a concomitant significant decrease in ( A ″) S- and ( A ‴) G2/M-phases was observed. The experiment was performed using biological quadruplicates. * p < 0.05; ** p < 0.01; ns: not significant; unpaired t test.

Article Snippet: The following primary antibodies and dilutions were used: CHRDL1 (#AF1808, R&D Systems, Wiesbaden-Nordenstadt, Germany) 1:2000; GAPDH (#CB1001, Calbiochem, Darmstadt, Germany) 1:20,000; OLIG2 (#AF2418, R&D Biosystems) 1:5000; SOX2 (#MAB2018, R&D Systems) 1:5000; SOX9 (#ab185966, Abcam, Cambridge, UK); SMAD1 [(#6944, Cell Signaling Technology (CST), Danvers, MA, USA)] 1:1000; SMAD5 [(#12534, CST)] 1:1000; pSMAD1/5 [(#9516, CST)] 1:1000.

Techniques: Transduction, shRNA

Journal: Cells

Article Title: CHRDL1 Regulates Stemness in Glioma Stem-like Cells

doi: 10.3390/cells11233917

Figure Lengend Snippet: Point-plots of Taqman-based gene expression of NCH644 GSCs after stable transduction with a non-targeting shRNA (shCtrl) or specific shRNA targeting CHRDL1 (shCHRDL1) and measurement of ( A ) CHRDL1 , ( B ) OLIG2 , ( C ) SOX2 , ( D ) SOX9 , ( E ) ZEB1 , ( F ) ZEB2 , ( G ) CD44 , ( H ) NES , ( I ) GFAP , ( J ) NEFL , ( K ) MAP2 and ( L ) RBFOX3 expression. The data are the summary of three experiments performed in triplicates. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001; ns: not significant; unpaired t test with Welch’s correction; white dot: shCtrl, red dot: shCHRDL1.

Article Snippet: The following primary antibodies and dilutions were used: CHRDL1 (#AF1808, R&D Systems, Wiesbaden-Nordenstadt, Germany) 1:2000; GAPDH (#CB1001, Calbiochem, Darmstadt, Germany) 1:20,000; OLIG2 (#AF2418, R&D Biosystems) 1:5000; SOX2 (#MAB2018, R&D Systems) 1:5000; SOX9 (#ab185966, Abcam, Cambridge, UK); SMAD1 [(#6944, Cell Signaling Technology (CST), Danvers, MA, USA)] 1:1000; SMAD5 [(#12534, CST)] 1:1000; pSMAD1/5 [(#9516, CST)] 1:1000.

Techniques: Gene Expression, Transduction, shRNA, Expressing

Journal: Cells

Article Title: CHRDL1 Regulates Stemness in Glioma Stem-like Cells

doi: 10.3390/cells11233917

Figure Lengend Snippet: Point-plots of Taqman-based gene expression of GS-5 GSCs after stable transduction with a non-targeting shRNA (shCtrl) or specific shRNA targeting CHRDL1 (shCHRDL1) and measurement of ( A ) CHRDL1 , ( B ) OLIG2 , ( C ) SOX2 , ( D ) SOX9 , ( E ) ZEB1 , ( F ) ZEB2 , ( G ) CD44 , ( H ) NES , ( I ) GFAP , ( J ) NEFL , ( K ) MAP2 and ( L ) RBFOX3 expression. The data are the summary of three experiments performed in triplicates. * p < 0.05; *** p < 0.001; **** p < 0.0001; ns: not significant; unpaired t test with Welch’s correction; white dot: shCtrl, red dot: shCHRDL1.

Article Snippet: The following primary antibodies and dilutions were used: CHRDL1 (#AF1808, R&D Systems, Wiesbaden-Nordenstadt, Germany) 1:2000; GAPDH (#CB1001, Calbiochem, Darmstadt, Germany) 1:20,000; OLIG2 (#AF2418, R&D Biosystems) 1:5000; SOX2 (#MAB2018, R&D Systems) 1:5000; SOX9 (#ab185966, Abcam, Cambridge, UK); SMAD1 [(#6944, Cell Signaling Technology (CST), Danvers, MA, USA)] 1:1000; SMAD5 [(#12534, CST)] 1:1000; pSMAD1/5 [(#9516, CST)] 1:1000.

Techniques: Gene Expression, Transduction, shRNA, Expressing

Journal: Cells

Article Title: CHRDL1 Regulates Stemness in Glioma Stem-like Cells

doi: 10.3390/cells11233917

Figure Lengend Snippet: CHRDL1-depletion reduces stemness-associated marker protein expression. Western Blots of GS-5 GSCs after stable transduction with a non-targeting shRNA (shCtrl) or specific shRNA targeting CHRDL1 (shCHRDL1) and measurement of the protein expression of OLIG2, SOX9, and SOX2. CHRDL1 was detected to validate efficient depletion and GAPDH served as a housekeeping protein.

Article Snippet: The following primary antibodies and dilutions were used: CHRDL1 (#AF1808, R&D Systems, Wiesbaden-Nordenstadt, Germany) 1:2000; GAPDH (#CB1001, Calbiochem, Darmstadt, Germany) 1:20,000; OLIG2 (#AF2418, R&D Biosystems) 1:5000; SOX2 (#MAB2018, R&D Systems) 1:5000; SOX9 (#ab185966, Abcam, Cambridge, UK); SMAD1 [(#6944, Cell Signaling Technology (CST), Danvers, MA, USA)] 1:1000; SMAD5 [(#12534, CST)] 1:1000; pSMAD1/5 [(#9516, CST)] 1:1000.

Techniques: Marker, Expressing, Western Blot, Transduction, shRNA

Journal: Cells

Article Title: CHRDL1 Regulates Stemness in Glioma Stem-like Cells

doi: 10.3390/cells11233917

Figure Lengend Snippet: CHRDL1-depletion activates BMP4-signaling. ( A , B ) Western Blot of ( A ) NCH644 treated with 20 ng/mL recombinant human BMP4 for 24 or 48 h or ( B ) NCH644 shCtrl and shCHRDL1. ( C , D ) Sphere formation assay of ( C ) NCH644 and ( D ) GS-5 shCtrl or shCHRDL1 GSCs and treatment with 25 ng/mL recombinant BMP4 or solvent for 7 days after seeding of 500 and 1000 cells per well. * p < 0.05; **** p < 0.0001; two-way-ANOVA with Sidak’s multiple comparison tests; white dot: shCtrl, red dot: shCHRDL1.

Article Snippet: The following primary antibodies and dilutions were used: CHRDL1 (#AF1808, R&D Systems, Wiesbaden-Nordenstadt, Germany) 1:2000; GAPDH (#CB1001, Calbiochem, Darmstadt, Germany) 1:20,000; OLIG2 (#AF2418, R&D Biosystems) 1:5000; SOX2 (#MAB2018, R&D Systems) 1:5000; SOX9 (#ab185966, Abcam, Cambridge, UK); SMAD1 [(#6944, Cell Signaling Technology (CST), Danvers, MA, USA)] 1:1000; SMAD5 [(#12534, CST)] 1:1000; pSMAD1/5 [(#9516, CST)] 1:1000.

Techniques: Western Blot, Recombinant, Tube Formation Assay, Solvent, Comparison

Journal: Cells

Article Title: CHRDL1 Regulates Stemness in Glioma Stem-like Cells

doi: 10.3390/cells11233917

Figure Lengend Snippet: CHRDL1-depletion radiosensitizes GSCs. ( A ) 53BP1 foci assay 24 h after irradiation of NCH644 shCtrl or NCH644 shCHRDL1 GSCs with the indicated doses. ( B ) Sphere formation of NCH644 shCtrl or shCHRDL1 cells 7 days after seeding of single cells and irradiation as indicated. ( C ) Representative microphotographs of NCH644 shCtrl or NCH644 shCRHDL1 without irradiation (0 Gy) or after irradiation with 6 Gy; scale bar: 200 µm. * p < 0.05; ** p < 0.01; **** p < 0.0001; two-way-ANOVA with Sidak’s multiple comparison tests. ( D ) Sphere formation of NCH644 treated with solvent or recombinant human BMP4 (25 ng/mL) immediately prior to irradiation with a dose of 4 or 6 Gy. Sphere area was determined 7 days after treatment. * p < 0.05; **** p < 0.0001; Kruskal—Wallis test with Dunn’s multiple comparisons test.

Article Snippet: The following primary antibodies and dilutions were used: CHRDL1 (#AF1808, R&D Systems, Wiesbaden-Nordenstadt, Germany) 1:2000; GAPDH (#CB1001, Calbiochem, Darmstadt, Germany) 1:20,000; OLIG2 (#AF2418, R&D Biosystems) 1:5000; SOX2 (#MAB2018, R&D Systems) 1:5000; SOX9 (#ab185966, Abcam, Cambridge, UK); SMAD1 [(#6944, Cell Signaling Technology (CST), Danvers, MA, USA)] 1:1000; SMAD5 [(#12534, CST)] 1:1000; pSMAD1/5 [(#9516, CST)] 1:1000.

Techniques: Irradiation, Comparison, Solvent, Recombinant

Journal: Cells

Article Title: CHRDL1 Regulates Stemness in Glioma Stem-like Cells

doi: 10.3390/cells11233917

Figure Lengend Snippet: CHRDL1 is associated with worse survival in glioma and ( A ) An unfavourable prognosis in ( B ) urothelial and ( C ) renal cancer. According to data from the human protein atlas (proteinatlas.org). * p < 0.05; *** p < 0.001; log-rank p -value for Kaplan–Meier plot. Image Credit: Human Protein Atlas.

Article Snippet: The following primary antibodies and dilutions were used: CHRDL1 (#AF1808, R&D Systems, Wiesbaden-Nordenstadt, Germany) 1:2000; GAPDH (#CB1001, Calbiochem, Darmstadt, Germany) 1:20,000; OLIG2 (#AF2418, R&D Biosystems) 1:5000; SOX2 (#MAB2018, R&D Systems) 1:5000; SOX9 (#ab185966, Abcam, Cambridge, UK); SMAD1 [(#6944, Cell Signaling Technology (CST), Danvers, MA, USA)] 1:1000; SMAD5 [(#12534, CST)] 1:1000; pSMAD1/5 [(#9516, CST)] 1:1000.

Techniques:

Journal: Nature Communications

Article Title: Organizer-derived Bmp2 is required for the formation of a correct Bmp activity gradient during embryonic development

doi: 10.1038/ncomms4766

Figure Lengend Snippet: ( a – c ) Chd protein distribution detected by confocal microscopy at different stages. Embryos injected with Tg(−2.067gsc:tBr-gfp) or Tg(−2.272bmp2b:tBr-gfp) DNA were harvested along with uninjected embryos at indicated stages for immunostaining using an anti-Chordin antibody and DAPI staining. Representative z-stack images were shown in animal-pole view with dorsal to the right. ( d – g ) The average ratios ( e – g ) of Chd to DAPI signals at the ventralmost or dorsalmost areas on optical equatorial sections at the blastodermal margin as illustrated in d . The strategy for estimating relative intensity was the same as described in the legend of . For each group, the average relative immunostaining intensity was calculated from 3 to 5 embryos and three optical sections at and nearby the blastodermal margin per embryo. The error bars represented s.d. The Chd/DAPI ratio in the ventralmost region at each stage was set to 1.0. Statistical significance (Student’s t -test): NS, nonsignificant ( P >0.05); * P <0.05; ** P <0.01. ( h – k ) chd transcripts distribution detected by in situ hybridization at indicated stages. Representative embryos were animal-pole views with dorsal to the right. The ratio of embryos with representative pattern was indicated for those shown in h – j ( l ) The ratio of embryos showing an expansion of chd expression at the shield stage (as shown in k ) n , number of observed embryos.

Article Snippet: The used antibodies for immunofluorescence were as follows: rabbit anti-zebrafish Bmp2b(NT) antibody (Anaspec, 1:50 diluted), rabbit anti-zebrafish Chordin (GeneTex, 1:50 diluted), mouse anti-Smad1 antibody (Santa Cruz), rabbit anti-phospho-Smad1/5/8 (Cell Signaling, 1:100 diluted), mouse anti-β-catenin antibody (Cell signaling, 1:100 diluted), sheep anti-Digoxigenin antibody (Roche, 1:4,000 diluted), DyLight 488-conjugated AffiniPure Goat anti-mouse IgG and DyLight 549-conjugated AffiniPure goat anti-rabbit IgG secondary antibodies (Jackson ImmunoResearch, 1:100 diluted).

Techniques: Confocal Microscopy, Injection, Immunostaining, Staining, In Situ Hybridization, Expressing