Journal: Cell Death & Disease
Article Title: Vitamin D opposes multilineage cell differentiation induced by Notch inhibition and BMP4 pathway activation in human colon organoids
doi: 10.1038/s41419-024-06680-z
Figure Lengend Snippet: A Light microscopy images of organoids (patient #110) cultured for 72 h in DIFF, BMP4, DBZ or B + D media in the absence (vehicle) or presence of calcitriol (100 nM). Scale bar, 500 μm. B RT-qPCR analysis of the RNA levels of enterocytic ( KRT20 , CA1 , KLF4 and FABP2 ) and mucosecretory ( AGR2 , TFF2 and TFF3 ) genes in organoids from patients #47, #86, #110, #130 for 48 h and #47, #110, #130, #159 for 72 h cultured in DIFF (red), BMP4 (blue), DBZ (orange) or B + D (green) media in the absence (vehicle) or presence of calcitriol (100 nM). * P < 0.05, ** P < 0.01, *** P < 0.001. C RT-qPCR analysis of the RNA levels of stemness genes in organoids from the same patients and in the same conditions as in B. D Western blot analysis and quantification of the effect of calcitriol on the expression of enterocytic (CA1) and mucosecretory (TFF2) marker proteins in organoids from four patients incubated in PROL or B + D media in the absence (vehicle) or presence of calcitriol (100 nM) for 48 h. The stemness PTK7 protein was used as control of differentiation and GAPDH as loading control. * P < 0.05. E Immunofluorescence analysis of the expression of enterocytic and mucosecretory protein markers in organoids (patient #158) cultured for 48 h in PROL or B + D media in the absence (vehicle) or presence of calcitriol (100 nM). Scale bars, 30 µm. Box-plots show the quantification of fluorescence intensity (Log 2 MGV) (patients #92, #158 and #166; the number of organoids analyzed was 68 for KLF4, 104 for KRT19, 110 for AGR2 and 79 for TFF2). ** P < 0.01, *** P < 0.001. F Western blot analysis and quantification of the level of phospho(P)-SMAD1/5/8 in organoid cultures that were incubated for 24 h with calcitriol (100 nM) or vehicle before incubation in DIFF medium or BMP4 medium (50 ng/mL) during the indicated times. MGV: mean gray value.
Article Snippet: Whole-cell extracts (15–30 μg or 80 μg the case of phospho-SMAD analyses) were separated by SDS-PAGE, transferred to PVDF membranes and incubated with the following primary antibodies: rabbit polyclonal-CA1 (CUSABIO, TX, USA, #CSBPA004364GA01HU), mouse monoclonal-GAPDH (Abcam, Cambridge, UK, #ab8245), rabbit monoclonal-PTK7 (Cell Signalling, MA, USA, #25618), rabbit polyclonal-SMAD1/5/8 (Santa Cruz, TX, USA, #sc6031-R), rabbit monclonal-Phospho-SMAD1/5/8 (Cell Signalling, #9511 S) and rabbit polyclonal-TFF2 (Proteintech, IL, USA, #13681-1-AP).
Techniques: Light Microscopy, Cell Culture, Quantitative RT-PCR, Western Blot, Expressing, Marker, Incubation, Control, Immunofluorescence, Fluorescence
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