1. A Bifunctional Approach of Immunostimulation and uPAR Inhibition Shows Potent Antitumor Activity in Melanoma 
Fanny Matheis, Markus V. Heppt, Saskia A. Graf, Peter Düwell, Claudia Kammerbauer, Achim Aigner, Robert Besch, Carola Berking 
Journal of Investigative Dermatology 
Volume 136, Issue 12, Pages (December 2016)
DOI: /j.jid
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2. Figure 1
Bifunctional ppp-uPAR generated by in vitro transcription combines uPAR gene silencing with RIG-I activation. (a) The specificity of two different ppp-uPAR oligonucleotides was assessed with quantitative real-time reverse transcriptase–PCR in 1205Lu melanoma cells, fibroblasts, and melanocytes 12 hours after transfection. (b) Induction of IFN-β 12 hours after transfection assessed with quantitative real-time reverse transcriptase–PCR. (c) Induction of IFN-β in culture supernatants of 1205Lu melanoma cells 12 hours after transfection (ELISA). Means ± standard deviations of three independent experiments are shown (a: Student t test; b, c: analysis of variance; ∗P < 0.05, ∗∗P < 0.01). (d) Immunoblot analysis of uPAR in 1205Lu (left) and fibroblasts (right) 72 hours after treatment. Bands or smears in uPAR immunoblots correspond to different glycosylated forms of uPAR. Detection of β-actin served as loading control. (e) Generation of ppp-uPAR by in vitro transcription. A, adenine; C, cytosine; ctrl, control; G, guanine; IVT, in vitro transcription; ns, not significant; OH, unmodified siRNA; rel, relative; ppp-ctrl, non-silencing 5′ triphosphate-conjugated small interfering RNA; ppp-uPAR, 5′ triphosphate-conjugated small interfering RNAs silencing uPAR; RIG-I, retinoic acid-inducible gene I; T, thymine; uPAR, urokinase-type plasminogen activator receptor.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
Copyright © 2016 The Authors Terms and Conditions

3. Figure 2
ppp-uPAR specifically reduces melanoma cell viability, whereas primary human cells are less sensitive. Viability was analyzed 16 hours, 72 hours, and 144 hours after transfection in (a) 1205Lu and (b) fibroblasts and (c) after 16 hours and 72 hours in melanocytes. The viability of OH-ctrl–treated cells was set to 100% (analysis of variance). (d) Cell viability and (e) death after 72 hours were less pronounced in 1205Lu after the IFN-α/β receptor was blocked. Isotype goat IgG served as control (Student t test). (f) Immunoblot analysis of (left) p53 and (right) Noxa and Puma 72 hours after transfection in 1205Lu cells. (g) p53 activity ELISA (left) 48 hours and (right) 72 hours after treatment of 1205Lu cells. Relative units compared with OH-ctrl are depicted (Student t test; ∗P < 0.05, ∗∗P < 0.01). ctrl, control; h, hours; ns, not significant; OH, unmodified siRNA; ppp-ctrl, non-silencing 5′ triphosphate-conjugated small interfering RNA; ppp-uPAR, 5′ triphosphate-conjugated small interfering RNAs silencing uPAR; rel., relative; uPAR, urokinase-type plasminogen activator receptor.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
Copyright © 2016 The Authors Terms and Conditions

4. Figure 3
Treatment with ppp-uPAR exerts pro-apoptotic effects in melanoma cells with acquired resistance to B-RAFi and MEKi. (a, c) Cell viability in WM239A resistant to (a) B-RAFi and (c) B-RAFi and MEKi. (b, d) Immunoblot analyses of uPAR, p53, and Noxa 72 hours after transfection in (b) WM239R and (d)WM239ADR. Parental WM239 without therapy resistance were included as control. (e, g) Cell viability in WM9 resistant to (e) B-RAFi and to (g) BRAFi and MEKi. (f, h) Immunoblot analyses of uPAR, p53, and Noxa 72 hours after transfection in (f) WM9R and (h) WM9DR. Parental WM239 without therapy resistance were included as control. The viability of OH-ctrl–treated cells was set to 100% in a, c, e, and g (analysis of variance, ∗∗P < 0.01). B-RAFi, B-RAF inhibition; ctrl, control; DR, double resistant; h, hours; MEKi, mitogen-activated protein kinase/extracellular signal-regulated kinase inhibition; ns, not significant; OH, unmodified siRNA; ppp-uPAR, 5′ triphosphate-conjugated small interfering RNAs silencing uPAR; R, single resistant; uPAR, urokinase-type plasminogen activator receptor.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
Copyright © 2016 The Authors Terms and Conditions

5. Figure 4
Treatment of tumor-bearing nude mice reduces melanoma growth and triggers a systemic immune response in vivo. Subcutaneous 1205Lu xenografts were generated in Fox n1nu/Fox n1nu mice. Mice were randomized into treatment groups, and treatment was started on day 8. Fifty μg RNA complexed with in vivo jetPEI (Polyplus, Ismaning, Germany) were injected intraperitoneally twice weekly. (a) Growth curves from pooled data of three mice per group are shown as means ± standard error of the mean (analysis of variance for each time point, ∗P < 0.05). (b) Representative image of two mice treated with ppp-uPAR2 or phosphate buffered saline (untreated) after being killed on day 27. Scale bars = 1 cm. (c) Serum levels of CXCL10 (ELISA). Blood was drawn 6 hours after the sixth RNA injection (day 27). Pooled data of two mice per group are shown as means ± standard deviation (analysis of variance, ∗∗P < 0.01). ctrl, control; CXCL10, C-X-C motif chemokine 10; ns, not significant; OH, unmodified siRNA; ppp-ctrl, non-silencing 5′ triphosphate-conjugated small interfering RNA; ppp-uPAR, 5′ triphosphate-conjugated small interfering RNAs silencing uPAR; uPAR, urokinase-type plasminogen activator receptor.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
Copyright © 2016 The Authors Terms and Conditions