Journal: Scientific Reports

Article Title: Inhibition of renalase expression and signaling has antitumor activity in pancreatic cancer

doi: 10.1038/srep22996

Figure Lengend Snippet: ( A ) Transient transfection of Panc1 cells using a RNLS-specific siRNA, or a non-specific control siRNA, and cell viability assayed 96 hrs later using the WST-1 reagent; n = 6, **indicates p < 0.001. ( B ) Cells were treated with indicated antibodies for 72 hrs and cell viability determined using WST-1; m28-RNLS and m37-RNLS: monoclonal antibodies raised against RNLS peptide RP220, ab31291: Abcam polyclonal antibody raised against a partial sequence of RP-220; n = 6, *indicates p < 0.005. ( C ) Representative photos of MiaPaCa2 cells after 3 days incubation with m28-RNLS, n = 10. ( D ) Athymic nude mice received subcutaneous injection of Panc1 cells transduced with RNLS shRNA (sh-RNLS) or control (sh-Control); tumor volume measured every 23 days for up to 30 days, n = 6 each; *indicates p < 0.05. ( E ) Nude mice xenografted with BxPC3, tumor volume measured prior to treatment every 3–4 days with 2 mg/kg of either rabbit IgG as a negative control or with m28-RNLS, n = 14, *indicates p < 0.05.

Article Snippet: To generate a stably transfected Panc1 cell line, cells were transduced with lentivirus (Santa Cruz) carrying either RNLS shRNA (sh-RNLS) or control shRNA (sh-Control) according to the manufacturer’s protocol.

Techniques: Transfection, Sequencing, Incubation, Injection, Transduction, shRNA, Negative Control

Journal: Scientific Reports

Article Title: Inhibition of renalase expression and signaling has antitumor activity in pancreatic cancer

doi: 10.1038/srep22996

Figure Lengend Snippet: ( A ) Representative images of sections from BxPC3 xenografted tumors (n = 14 each) treated with anti-m28-RNLS or control rabbit IgG, and of xenografts of Panc1 cells transduced with RNLS shRNA (sh-RNLS) or control (sh-Control). Tissues are stained for TUNEL (arrows: positive cells), cell proliferation marker Ki67 (brownish stain), and cell cycle inhibitor p21 (brownish stain). ( B ) FACS analysis of Panc1 cells in culture treated with either m28-RNLS (30 μg/ml) or 100 μM etoposide (positive control) for 4 days; n = 3, *indicates p < 0.05. ( C ) Effect of m28-RNLS on cell cycle of Panc1 cells determined by FACS analysis; green curve: no treatment, purple curve: rabbit IgG, red curve: m28-RNLS 30 μg/ml. (D ) Panc1 cells treated with polyclonal ab31291 or with goat IgG as a negative control, and cell lysates probed for activation of p38 by western blot from day 0 to 4. Representative blot, n = 4.

Article Snippet: To generate a stably transfected Panc1 cell line, cells were transduced with lentivirus (Santa Cruz) carrying either RNLS shRNA (sh-RNLS) or control shRNA (sh-Control) according to the manufacturer’s protocol.

Techniques: Transduction, shRNA, Staining, TUNEL Assay, Marker, Positive Control, Negative Control, Activation Assay, Western Blot

Journal: Scientific Reports

Article Title: Inhibition of renalase expression and signaling has antitumor activity in pancreatic cancer

doi: 10.1038/srep22996

Figure Lengend Snippet: ( A ) Activation of STAT3 by RNLS in Panc1 cells; Panc1 cells in culture treated with either BSA or RNLS, and STAT3 phosphorylation assessed by western blot; p-Ser 727 -STAT3: phosphorylation at serine 727, p-Y 705 -STAT3: phosphorylation at tyrosine 705; representative study. ( B ) Quantification of STAT3 phosphorylation with RNLS; signals normalized to total STAT3; n = 3, *indicates p < 0.05. ( C ) PDAC lines BxPC3, Panc1 and MiaPaCa2 are serum starved for 48 hrs, then incubated with 30 μg/ml of either bovine serum albumin (BSA) or rRNLS for 3 days; total and live cell number determined using trypan blue and an automated cell counter; n = 4, **indicates p < 0.0001. ( D ) Effect of rRNLS on survival of Panc1 cells in the absence or presence of 10 μM U0126 (Erk inhibitor) or 50 μM AG490, (JAK2 and STAT3) for 72 h. The doses of AG490 and U0126 chosen had been reported to inhibit the phosphorylation of JAK and ERK1/2 respectively in Panc1 cells. The time point of 72 hrs was chosen to allow the cell time to proliferate. Cell proliferation was measured by the WST-1 method, and depicted as % change of DMSO-treated Panc1 cells. n = 6, *=p < 0.01. ( E ) siRNA mediated inhibition of PMCA4b expression blocks RNLS mediated MAPK signaling; Left and middle panels : MiaPaCa2 cells transfected with either non-targeting or PMCA4b siRNA, maintained in serum free medium for 3 days and treated with either 25 μg of BSA or 25 μg of RNLS peptide RP-220 for the indicated time; RP-220 mediated ERK and STAT3 activation assessed by western blot and representative immunoblots are shown; p-ERK = phosphorylated ERK, p-Y 705 -STAT3 = phosphorylated STAT3, p-S727-STAT3 = phosphorylated STAT3, BSA = bovine serum albumin, RP-220 = RNLS peptide agonist; Right panel : quantification of phosphorylated ERK (p-ERK), signals normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) loading control; n = 4, *=P < 0.03, **=p < 0.0001.

Article Snippet: To generate a stably transfected Panc1 cell line, cells were transduced with lentivirus (Santa Cruz) carrying either RNLS shRNA (sh-RNLS) or control shRNA (sh-Control) according to the manufacturer’s protocol.

Techniques: Activation Assay, Western Blot, Incubation, Inhibition, Expressing, Transfection