Journal: eLife

Article Title: Increased expression of heme-binding protein 1 early in Alzheimer's disease is linked to neurotoxicity

doi: 10.7554/elife.47498

Figure Lengend Snippet: Figure 4. Analysis of Hebp1 expression in the brain of 3Tg-AD mice. (A) Expression of Hebp1 in 12-month-old control and 3Tg-AD mice by brain region. (B) Hebp1 immunostaining of the fronto-temporal cortex depicting primary motor and somatosensory areas and hippocampus (coronal sections). CA1 region is marked with the white dashed line. (C) Co-staining of Hebp1 with markers of CA1 and dentate gyrus neurons (Ctip2), astrocytes (GFAP) and microglia (IBA-1) in the hippocampus of 3Tg-AD mice. Hepb1 is expressed predominantly in Ctip2-positive cells of hippocampus (neurons). All images were acquired from 12-month-old control or 3Tg-AD mice. Scale bar is 100 mm. All data shown are representative of results obtained from three independent experiments. DOI: https://doi.org/10.7554/eLife.47498.012 The following figure supplements are available for figure 4:

Article Snippet: The following primary antibodies were used for immunoblotting: rabbit Hebp1 (1:1000, Invitrogen, PA5-30609), mouse Glo1 (1:1000, GeneTex, Irvine, CA, GTX628890), rabbit CA1 (1:250, Novus Biologicals, Abingdon, UK, NBP1-88191), mouse a-tubulin (1:5000, Synaptic Systems, Göttingen, Germany, 302 211), rabbit b-actin (1:5000, Synaptic Systems, 251 003), rabbit GFP (1:5000, Synaptic Systems, 132 002), mouse Rab5 (1:1000, Synaptic Systems, 108 111), rabbit Rab6 (1:1000, Synaptic Systems, 273 003), rabbit Lamp1 (1:500, Abcam, ab24170), mouse Mic60 (1:1000, Abcam, ab110329), rabbit Cox4 (1:1000, Synaptic Systems, 298 002), rabbit CytC (1:1000, Cell Signaling, Beverly, MA, USA, 11940S), rabbit caspase 9 (1:1000, Abcam, Cambridge, UK, ab185719), mouse Sodium Potassium ATPase, subunit a1 (1:1000, Abcam, Cambridge, UK, ab7671), mouse syntaxin 1 (1:1000, Synaptic Systems, 110 001), mouse VAMP2 (1:10000, Synaptic Systems, 104 211), rabbit phospho-tau (Ser400;Thr403;Ser404) (1:1000, Cell Signaling, Beverly, MA, USA, 11837S).

Techniques: Expressing, Control, Immunostaining, Staining

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