Figure 6
From: In vitro evidence for senescent multinucleated melanocytes as a source for tumor-initiating cells
OIS is required for the pluripotent phenotype. (a) Phase-contrast (PH) images of unstained Hmhi cells after 14, 21 and 35 days of EGF treatment (100 ng/ml) and brightfield images of SA-β-galactosidase-stained (β-Gal) Hmhi cells after 14 days of EGF treatment. (b) Nanog-driven GFP expression (GFP) in Hmhi and Hmhi-AR cells, transiently transfected with the PL-SIN-EOS-S(4+)-eGFP vector. Scale bars, 100 μm. (c) Phase-contrast images of melan-a cells cultivated for 28 days without any additives (−) or in the presence of TPA. Scale bars, 100 μm. (d) N-RAS61K cells were cultivated for 28 days in the presence of doxycycline (Dox), whereas melan-a cells were kept in media with or without TPA. Upon 28 days of treatment, supernatant was transferred to a new 6-well plate, which was incubated for 24 h to allow cells to re-attach, followed by a 2% crystal violet staining. (e) Left panel: Real-time PCR analysis of Tyrp1, Dct and Mlana from melan-a cells cultivated for 28 days in the absence or presence of TPA, respectively. (e) Right panel: RT-PCR analysis of Nanog, Spo11 (40 cycles each) and Pdpn (33 cycles) expression of melan-a cells starved for 28 days or treated with TPA, respectively. Dox-treated N-RAS61K cells, also harvested after 28 days, served as a positive control. Hprt was used as a reference. *P<0.05
