1. SIRT1 Regulates Lamellipodium Extension and Migration of Melanoma Cells 
Risa Kunimoto, Kowichi Jimbow, Akihiko Tanimura, Masahiro Sato, Kouhei Horimoto, Takashi Hayashi, Shin Hisahara, Toshiya Sugino, Tomohisa Hirobe, Toshiharu Yamashita, Yoshiyuki Horio 
Journal of Investigative Dermatology 
Volume 134, Issue 6, Pages (June 2014)
DOI: /jid
Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

2. Figure 1
Cytoplasmic SIRT1 promotes the migration of B16F1 cells. (a) Immunostaining of SIRT1 in melanoma cells and melanoblasts. Bars=10 μm. (b) Reverse trancriptase–PCR (RT-PCR) and western blot analysis of SIRT1 in melanoma cells. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control. (c) Expression of SIRT1 in the cytoplasmic fraction of B16F1 cells. Lamin A/C and cytosolic aspartate aminotransferase (cAAT) were markers of the nucleus and cytoplasm, respectively. (d, e) SIRT1 modulators affected B16F1 cell migration in wound-healing assay (d) and transwell migration assay (e). (f) SIRT1 small interfering RNAs (SIRT1-siRNAs) suppressed SIRT1 mRNA expression. (g) Time course of SIRT1 protein expression levels in B16F1 cells after knockdown by si1-1. (h) SIRT1–siRNAs suppressed the cell migration of B16F1 cells. Data from at least three independent experiments are represented as means±SD. *P<0.05, **P<0.005; NS, no significance.
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

3. Figure 2
SIRT1 is necessary for lamellipodium extension of B16F1 cells. (a) Representative images of SIRT1 and phalloidin in the absence (upper panels) or presence (lower panels) of serum. Merged images were also shown. Serum induced a lamellipodium in which SIRT1 existed. The binding of SIRT1 with cortactin (right panels). (b, c) Representative images of phalloidin and Hoechst in B16F1 cells expressing control-siRNA (c-si) or SIRT1-siRNA (si1-1) and quantification of lamellipodium extension. (d, e) Effect of nicotinamide (NAM) on platelet-derived growth factor (PDGF)-induced lamellipodium extension. Representative images of pAkt, phalloidin, and cortactin, and quantification of lamellipodium extension. (f, g) Effects of NAM and LY on Rac-induced lamellipodium extension. Cells were stimulated by 20% serum for 20 minutes. Representative images of EGFP, phalloidin, and Hoechst in cells expressing enhanced green fluorescent protein (EGFP) alone (control), RacV12+EGFP, or RacN17+EGFP and quantification of lamellipodium extension. White circles indicate lamellipodia. Data from at least three independent experiments are represented as means±SD. *P<0.05 and **P<0.005; NS, no significance. Bar=10 μm.
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

4. Figure 3
Nicotinamide (NAM) affects platelet-derived growth factor (PDGF)-induced phosphatidylinositol-3,4,5-trisphosphate (PIP3) level in B16F1 cells. (a–c) Effect of LY in a wound-healing assay (a) and lamellipodium formation by serum and PDGF (b, c). (b) Representative images of pAkt, phalloidin, and cortactin. White circles: lamellipodia. (c) Data from three independent experiments are represented as means±SD. *P<0.05, **P<0.005; NS, no significance. Bars=1 mm (a), 5 μm (b). (d) Time course of representative fluorescence resonance energy transfer images of PIP3 in cells expressing fllip-pm (upper and middle panels) and fllip-pm-R284C (lower panels). Middle panels show a cell treated with NAM. (e) Intensity of PIP3 fluorescence at the peripheral area (diamond) and at the central area (square) of upper panels of d. (f) Intensity of PIP3 fluorescence of middle panels of d. Arrowheads: PIP3 increase. Bar=10 μm. (g) Mean duration time of the increased PIP3. Data are represented as means±SD.
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

5. Figure 4
SIRT1 inhibition prevents the metastasis of B16F1 cells in vivo. (a) Abdominal invasion was compared using the χ2-test. (b) The Kaplan–Meier analysis of survival in nicotinamide (NAM)-treated mice (n=30) and control saline-treated mice (n=29). Significant lifespan extension in NAM-treated mice. Data were compared using the log-rank test. (c) The expression levels of SIRT1 mRNA in c4 and sh9 B16F1 cells. (d, e) Migration activity of sh9 cells was lower than that of c4 cells in wound-healing assays (d) and transwell assays (e). Bars=1 mm (d), 100 μm (e). (f) The abdominal invasion and/or lymph node metastasis of mice transplanted with c4 or sh9 cells were compared using the χ2-test. (g) Number of lung metastatic nodules of mice injected with c4 or sh9 cells. Data were compared with Welch’s t-test. (c–e) Data from at least three independent experiments are represented as means±SD. *P<0.05, **P<0.01; NS, no significance.
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions