Conserved (RBPJL: R220, F262, L393). These amino acids are highlighted in red in the major amino acid sequences (see Figure 1A). 3.2. Expression of RBPJL Is Extremely Particular and Overlaps with PTF1a We compared relative mRNA levels of RBPJL (Figure 2A,B) and RBPJ (Figure 2C,D) in distinctive tissues from Mus musculus and Homo sapiens by qRT-PCR. The expression of RBPJ is ubiquitous, also clearly detectable in human pancreatic tissue, PDAC and pancreatic cancer cell lines (Figure 2D). In contrast, RBPJL expression is highly expressed inside the pancreas in each mouse (Figure 2A) and human (Figure 2B). Surprisingly, in human PDAC samples RBPJL is significantly much less expressed in comparison with RBPJ (compare Figure 2B,D). Furthermore, RBPJL expression is almost undetectable in human PDAC cell lines. Considering that tumor cells resemble a Bioactive Compound Library manufacturer ductal fate in PDAC, we hypothesized that RBPJL not simply is actually a pancreas precise marker, but much more specifically, is an acinar marker of the pancreas. For that reason, we re-analyzed single-cell RNAseq data from human adult pancreas samples (GSE81547, [29]) with regard towards the expression on the two paralogs RBPJ and RBPJL. Once more, RBPJ is expressed in all subtypes of cells, which includes acinar-, ductal- and mesenchymal types (compare Figure S2A with Figure S2B). PTF1a is actually a wellknown acinar marker, and, when mapping RNA-levels in single cells, the overlap is clearly within the acinar fraction (upper left) plus a small amount within the progenitor fraction, see Figure S2C. The expression of RBPJL is practically identical to PTF1a expression (compare Figure S2C with Figure S2D). Additionally, when we applied a well-established acinar-toductal differentiation model ex vivo by adding TGF to freshly isolated and dissociated pancreata from wildtype mice, ductal differentiation is evident just after 3 days (Figure S3A, inlay at reduce correct). This acinar to ductal differentiation may be monitored by qRT-PCR showing the upregulation from the ductal marker cytokeratine 19 (Ck19) collectively with a downregulation from the acinar marker Ptf1a, amylase (Amy2a2) and once more Rbpjl (Figure S3B). Together, RBPJL expression is specifically restricted towards the pancreatic acinar lineage and strongly induced therein, whereas RBPJ is much more ubiquitously expressed.Cancers 2021, 13,9 ofFigure 1. Comparison of RBPJ and RBPJL: (A) Protein sequence CX-5461 Technical Information alignment of mouse RBPJ and mouse RBPJL. RBPJ consists of three domains: the NTD (N-terminal domain, cyan), the BTD (beta-trefoil domain, green), and the CTD (C-terminal domain, orange). The “linker region” among the BTD and the CTD is highlighted in magenta. The numbers indicate the amino acid positions. Residues inside RBPJ essential for DNA binding (R218) and SHARP binding (F261 and L388, highlighted in red) are conserved in between RBPJ and RBPJL. (B) Structural alignment of RBPJ and RBPJL in complex with DNA depending on homology modeling. Structure of RBPJ bound to DNA (left; PDB entry 3BRG), RBPJL bound to DNA (middle) and also the structural alignment of both complexes (proper) reveal a higher conservation on the structural level. The NTD, BTD and CTD of RBPJ are presented in the same color code as in (A). The putative homolog domains inside RBPJL are labeled in dark blue (NTD), dark green (BTD) and dark yellow (CTD). The linker area can also be colored in magenta. The DNA is colored in gray. Reduce panels show the complexes just after 90 rotation around a vertical axis revealing the accountable DNA binding regions of RBPJ and RBPJL. All structures, also because the align.