Fig 1: NISCH agonist rilmenidine reduces cell migration and invasion in vitro. (a) Quantification of the number of PDAC cells spread 6 and 24 h after seeding on tissue culture plastic, collagen I or fibronectin-coated surface in the presence of rilmenidine. Results were expressed as the percentage of spread cells out of the total counted per field. (b) Quantification of the PDAC cell migration in the wound healing assay after 24 h of treatment with rilmenidine; (c) Quantification of the PDAC cell migration in the transwell assay after 48 h of treatment with rilmenidine; (d) Level of active GTP-bound Rac1 in PANC-1 cells from the wound healing assay 24 h post wounding in control or 100 μM rilmenidine-treated PANC1 cells, pulled–down from 600 μg lysate treated with 10 μL PAK-PBD beads; 20 ng of His-tagged Rac1 protein control, positive cellular protein control GTPγS loaded lysate, negative cellular protein control GTP loaded lysate; (e) Quantification of PDAC cell invasion through collagen I, fibronectin or matrigel in the transwell assay after 72 h of treatment with rilmenidine; (f) Representative images of transwell invasion through matrigel coat (right). Scale bar 200 μm. All data are shown as mean ± SD. n = 3; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, two-way ANOVA (Sidak’s multiple comparisons test).
Fig 2: Nischarin is expressed in PDAC. (a) NISCH mRNA expression in normal and tumor pancreatic tissues from the TCGA PAAD cohort; (b) NISCH mRNA expression in the paired tumor and adjacent tissue samples in the GSE62452 cohort; (c) NISCH mRNA expression by stage in the TCGA PAAD cohort, ** p = 0.0052, *** p = 0.0003, adjusted p-value, one-way ANOVA, Tukey’s multiple comparison test, and (d) the GSE62452 cohort, * p = 0.0414, one-way ANOVA, Tukey’s multiple comparison test; (e) Kaplan-Meier plots for the overall and (f) progression free survival of PDAC patients in the TCGA PAAD cohort divided into two groups using the best cut-off value for NISCH. p = 0.01 and p = 0.018 by Mantel-Cox test, respectively; (g) The overall survival of patients in the GSE62452 cohort (p = 0.19 by Mantel-Cox test); (h) NISCH mRNA expression in groups based on the status of the 3p chromosome, in patients from the TCGA PAAD cohort, * p = 0.0299, one-way ANOVA, Tukey’s multiple comparison test; (i) Total NISCH protein level in normal and cancer pancreatic tissue from CPTAC; (j) NISCH expression in the normal and tumor pancreatic tissue from the NBP2-78128 microarray, scale bar 20 μm; (k) NISCH expression in the normal and tumor pancreatic tissue deposited in the Human Protein Atlas (HPA023198), scale bar 20 μm.
Fig 3: Effects of NISCH agonist treatment on PDAC cells in vitro. (a) Percent viability of PDAC cells after 72 h of treatment with increasing concentrations of rilmenidine, moxonidine or clonidine determined by MTT assay, presented as log(inhibitor) vs. normalized response. Experiment was performed in triplicates, with at least three biological repeats. (b) The proportion of live Annexin V-/PI- cells in the population of untreated and cells treated with 100 and 300 μM rilmenidine for 24 h, (c) 48 h, or (d) 72 h. Results are shown as % of viable (Annexin V-/PI-) cells; (e) Gene Ontology functional gene expression enrichment analysis of MIA PaCa-2 cells treated with 100 μM rilmenidine for 24 h or (f) 48 h.
Fig 4: Nischarin is expressed in both the epithelial and stromal compartments of PDAC. (a) NISCH mRNA expression in paired epithelial and stromal compartments of PDAC from the GSE93326 cohort, **** p < 0.0001, two-tailed paired t-test; (b) NISCH mRNA expression in the ECM-rich and immune-rich type stroma in the GSE93326 cohort, ns = non-significant (p ≥ 0.05); (c) NISCH (green) and FAP (red) localization in the normal and tumor pancreatic tissue from the NBP2-78128 microarray, nuclei blue; scale bar 100 μm (d) NISCH (green) and αSMA (red) localization in the normal and tumor pancreatic tissue from the NBP2-78128 microarray, nuclei blue; scale bar 100 μm (e) NISCH protein expression in PDAC cancer cell lines, and in (f) patient-derived CAFs.
Fig 5: Rilmenidine affects CAF phenotype (a) NISCH expression (green) in CAFs isolated from two patient tissues, phalloidin red, scale bar 20 μm; (b) percent viability of CAFs after 72 h of treatment with increasing concentrations of rilmenidine, determined by MTT assay, presented as log(inhibitor) vs. normalized response; (c) immunofluorescence staining of CAF markers α-SMA (red), FAP (red), collagen I (green) and fibronectin (green) after 72 h of rilmenidine treatment in CAF1 (left panel) and CAF2 (right panel) cells, scale bar 50 μm; (d) quantification of the immunofluorescence staining with Image J, expressed as percent area per nucleus for FN1 and COL1 and as integrated density per nucleus for α-SMA and FAP, for three CAF cell lines, normalized to the untreated condition. p-values from paired t-test; (e) expression of α-SMA, FAP and fibronectin in CAF1, CAF2 and CAF3 cell lysates, after 72 h of rilmenidine treatment; (f) quantification of western blots with Image J presented as integrated density of the select protein divided by the integrated density of the housekeeping protein normalized to the untreated condition. p-values from paired t-test.
Supplier Page from OriGene Technologies for NISCH Human shRNA Plasmid Kit (Locus ID 11188)