Fig 2: CHRNA7 is a major binding site for SLURP1 on PDAC cells(A) Simultaneous FACS and qRT-PCR analyses revealed CHRNA7-positivity of all eight tested PDAC cell lines, whereby membranous CHRNA7-staining showed significant inter-experimental variability and lack of correlation with RNA levels. The graph summarizes the data of two independent experiments shown below and measuring CHRNA7 mRNA (qRT-PCR; log10-transformed number of transcripts/10k PPIB) and surface protein expression (log10-transformed mean fluorescence intensity/MFI values) in unrelated passages of each cell line. (B) Individual FACS histograms illustrating intra- and inter-experimental variability are shown as an overlap of anti-CHRNA7-IgG (gray tinted area) and rabbit IgG (bold black line; isotype control) profiles; the difference in their geometric MFIs was used to calculate final MFI value for each culture. (C) qRT-PCR data demonstrating the inter-experimental stability of CHRNA7 mRNA expression also revealed the existence of the two groups, whereby CHRNA7high-group but not CHRNA7neg/low-group showed RNA-agreeable, stable pattern of protein expression. (D–F) The siRNA-based depletion of CHRNA7 confirmed the specificity of the reagents such as primers (qRT-PCR, D) and antibodies (Western blot, E, and FACS, F). As illustrated for COLO357 cultures, transfection with three commercially available CHRNA7-specific siRNA sets (si#1, si#2, si#3) eliminated over 75% of CHRNA7 mRNA and protein compared to the transfection of cells with negative control siRNA set (neg.si) or left untreated (control). (G) siRNA-based depletion of CHRNA7 indicated the preferential binding of SLURP1 to this receptor on PDAC cells. For comparison, FITC-conjugated SLURP1 was added to COLO357 and PANC-1 cells transfected with three different CHRNA7-siRNA sets (‘CHRNA7-depletion’ group depicted by grey-, green- and rose-colored profiles for si#1, si#2 and si#3, respectively) and to cells remaining intact or transfected with negative siRNA (‘controls’ group depicted by blue and lilac-colored profiles).

Fig 3: PAH is associated with an increase in RV Collagen and ACh.(A–C) Collagen (A), ACh (B), and a7 nAChR (C) protein expression assessed using Sircol assay, ELISA, and immunoblot, respectively, in explanted RV tissue from end-stage human PAH (n = 5) and controls (n = 4). (D) Immunofluorescent staining for a7 nAChR (green), vimentin (red), and nuclei (blue) in human RV tissue from control. Arrows demonstrate localization of a7 nAChR in CF (vimentin positive cells). Scale bar: 20 µm. Data are shown as mean ± SEM. Two-tailed t tests were performed between control and PAH. *P < 0.05. ACh, acetylcholine; nAChR, nicotinic acetylcholine receptor; PAH, pulmonary arterial hypertension; RV, right ventricle.

Fig 4: The relative effect of specific a7-nAChR antagonists to block ACh (10-5 M) elicited responses.A- aBgTx attenuated the ACh evoked contractions in a concentration dependent manner. The blocking effects of 50 nM and 100 nM toxin were statistically significant. The recovery is almost complete. B- The block by 50 nM aA-CTx PIVA was not significant, while the a-CTx ImI at 100 nM blocked almost completely the ACh elicited contraction. The recovery from block was partial. n = number of muscles. Asterisks indicate a significant difference from the control value: *P<0.01.

Fig 5: PH is associated with an increase in RV ACh/a7 nAChR.(A) ELISA for ACh performed on homogenates from the RV at 3 wks (n = 5 Con/6 PH) and 7 wks (n = 12 Con/12 PH) and LV at 3 wks and 7 wks (n = 6 Con/6 PH). (B) AChE activity measured in homogenates from the RV and LV at both time points (n = 6 Con/6 PH). (C) Expression of a7 nAChR protein relative to Vinculin in RV at 3 wks (n = 6 Con/6 PH) and 7 wks (n = 13 Con/12 PH) normalized to controls. (D) Expression of a7 nAChR mRNA in freshly isolated RVCF and RV cardiomyocytes from control and 7 wk PH animals (n = 6 Con/6 PH). (E) Quantitated immunofluorescence intensity of a7 nAChR in nonpermeabilized isolated RVCF from 7-wk control and PH animals. (F) Summary schematic. Data are shown as mean ± SEM. Two-tailed t tests were performed between control and PH at 3 and 7 wks; Mann-Whitney U test for real-time PCR data. *P < 0.05. ACh, acetylcholine; AChE, acetylcholinesterase; CF, cardiac fibroblasts; CM, cardiomyocytes; Glyceraldehyde 3-phosphate dehydrogenase; IF, immunofluorescence; nAChR, nicotinic acetylcholine receptor; PH, pulmonary hypertension; RV, right ventricle.