Conserved (RBPJL: R220, F262, L393). These amino acids are highlighted in red within the primary amino acid sequences (see Figure 1A). 3.2. Expression of RBPJL Is Highly Distinct and Overlaps with PTF1a We compared relative mRNA levels of RBPJL (Figure 2A,B) and RBPJ (Figure 2C,D) in distinct 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 extremely expressed in the pancreas in each mouse (Figure 2A) and human (Figure 2B). Surprisingly, in human PDAC samples RBPJL is substantially less expressed in comparison to RBPJ (examine Figure 2B,D). In addition, RBPJL expression is pretty much undetectable in human PDAC cell lines. Since tumor cells resemble a ductal fate in PDAC, we hypothesized that RBPJL not only can be a pancreas precise marker, but extra specifically, is an acinar marker from the pancreas. Thus, we re-analyzed single-cell RNAseq data from human adult pancreas samples (GSE81547, [29]) with regard towards the expression with the two paralogs RBPJ and RBPJL. Again, RBPJ is expressed in all subtypes of cells, including acinar-, ductal- and mesenchymal forms (evaluate Figure S2A with Figure S2B). PTF1a is often a wellknown acinar marker, and, when mapping RNA-levels in single cells, the overlap is clearly inside the acinar fraction (upper left) plus a smaller amount in the progenitor fraction, see Figure S2C. The expression of RBPJL is virtually identical to PTF1a expression (compare Figure S2C with Figure S2D). Additionally, when we utilized 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 soon after 3 days (Figure S3A, inlay at reduced appropriate). This acinar to ductal differentiation can be monitored by qRT-PCR showing the upregulation from the ductal marker cytokeratine 19 (Ck19) collectively having a downregulation on the acinar marker Ptf1a, amylase (Amy2a2) and once again Rbpjl (Figure S3B). With each other, RBPJL expression is particularly restricted for the pancreatic acinar lineage and strongly induced therein, whereas RBPJ is more ubiquitously expressed.Cancers 2021, 13,9 ofFigure 1. Comparison of RBPJ and RBPJL: (A) Protein sequence alignment of mouse RBPJ and mouse RBPJL. RBPJ consists of three domains: the NTD (N-terminal domain, cyan), the BTD (beta-trefoil domain, green), along with the CTD (C-terminal domain, orange). The “linker region” among the BTD and also the CTD is highlighted in magenta. The numbers Pitstop 2 medchemexpress indicate the amino acid positions. Residues inside RBPJ critical for DNA binding (R218) and SHARP binding (F261 and L388, highlighted in red) are conserved between RBPJ and RBPJL. (B) Structural alignment of RBPJ and RBPJL in complicated with DNA according to homology modeling. Structure of RBPJ bound to DNA (left; PDB entry 3BRG), RBPJL bound to DNA (middle) along with the structural alignment of both complexes (proper) reveal a higher conservation on the structural level. The NTD, BTD and CTD of RBPJ are presented inside the identical colour code as in (A). The putative homolog domains inside RBPJL are Diminazene Activator labeled in dark blue (NTD), dark green (BTD) and dark yellow (CTD). The linker region can also be colored in magenta. The DNA is colored in gray. Lower panels show the complexes after 90 rotation about a vertical axis revealing the responsible DNA binding regions of RBPJ and RBPJL. All structures, too because the align.