Journal: Scientific Reports
Article Title: Delta-like 1-mediated cis-inhibition of Jagged1/2 signalling inhibits differentiation of human epidermal cells in culture
doi: 10.1038/s41598-019-47232-2
Figure Lengend Snippet: Dynamic expression pattern of Notch signalling components in human keratinocyte cultures. ( a–c ) Heatmaps showing Log 2 fold change of normalised gene expression for pairwaise comparisons of mRNA levels of Notch receptors (Notch1(N1), Notch2(N2), Notch3(N3)) ( a ), Notch ligands (Delta-like1(Dll1), Jagged1(JAG1), Jagged2(JAG2) ( b ), and stem cell (SC)/terminal differentiation markers ( c ) during suspension culture. For each condition the mean of n = 3 independent replicates in the microarray dataset was used and the pairwise fold change comparison was between the means of both samples. ( d–h) Q-RT PCR analysis of mRNA levels of terminal differentiation markers ( d ), Notch signalling target genes ( e ) and Notch receptors and ligands ( f–h ) in enriched populations of stem- (SC), committed progenitor- (CP), and terminally differentiated (TD) cells. Data shown are from n = 5 independent experiments. Bars in ( d–g ) represent the average fold change in mRNA abundance (normalised to 18sRNA) compared to the SC-enriched fraction (red line) in each experiment. Bars in ( h ) represent mean ΔCq expression. Error bars represent S.D. P-values were calculated using one-way ANOVA with Holm Sidak’s multiple comparisons test (*p < 0.05). ( i ) Western blots of enriched keratinocyte populations (as in d–h ) using antibodies against the TMICD and NEXT fragments of Notch1, 2 or 3, the ICD of Notch1, integrin (ITG)β1, or involucrin (IVL). Tubulin was used as loading control. Numbers below lanes represent normalised protein ratios relative to an arbitrary level of 1.0 set for the SC-enriched fraction. Note that the Notch1 and Notch2 TMICDs and NEXT fragments cannot be separated sufficiently by SDS-PAGE to appear as discreet bands. ( j ) Western blots of keratinocytes cultured in KSFM or FAD, in the presence or absence of DAPT, using antibodies against the TMICD and NEXT fragment of Notch2, or IVL. GAPDH was used as loading control. Note accumulation of the Notch2 TMICD and NEXT fragment upon DAPT treatment in differentiating cells, indicating Notch2 receptor activation upon differentiation commitment. ( k ) Schematic illustration summarising expression patterns of different Notch receptors and ligands. ECM: extracellular matrix.
Article Snippet: Wells were incubated for 4 h at RT with recombinant Fc-tagged Notch ligand proteins Dll1 (Adipogen Life Science), Jagged1 and Jagged2 (R&D) or antibodies against the β2 microglobulin (β2MG) subunit of the human major histocompatibility complex (Abcam) (2.5 µg/cm 2 ), diluted in 0.1% (w/v) BSA in Hanks Buffered Salt Solution (HBSS).
Techniques: Expressing, Microarray, Reverse Transcription Polymerase Chain Reaction, Western Blot, SDS Page, Cell Culture, Activation Assay
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