Scr004

C33A and HaCaT cells were plated at a density 1x10⁵ and 3x10⁵ respectively for transfection experiments. 24-hours post-seeding the cells were subjected to transfection with Fugene transfection reagent. For the transduction experiments, 293T cells were used at a density of 1x10⁶. Lentiviral or retroviral constructs were used as mentioned in S2 Table [42 (link)–50 , 68 –69 (link)]. The transfection of 293T cells was performed using Xtreme9 transfection reagent. 48 hours post-transfection the lentivirus/retrovirus was collected from 293T cells and applied to HaCaT, HeLa, CaSki and C33A with 1ug/ml Polybrene. Stable cell lines generated by retroviral transductions were selected with specific antibiotics as mentioned in S2 Table. For the generation of iPs, MEFs were plated and transduced with retroviruses expressing pMXs-Sox2 pMXs-Klf4 and pMXs-Oct4 or the HPV viral oncogenes. Transduced MEFs were grown in iPS medium for a couple of weeks until iPS colonies formed. Alkaline phosphatase staining was performed on fixed colonies using the Alkaline phosphatase detection kit according to the manufacturer’s instructions (Millipore SCR004). Positive foci with a diameter of ≥1mm were counted.

Alkaline phosphatase (AP) activity was visualized by the commercial AP staining kit (Millipore #SCR004; Schwalbach, Germany) according to the manufacturer’s instructions. In order to characterize pluripotency of all AD-iPSC colonies, the cells were fixed in phosphate-buffered saline (PBS) containing 4% paraformaldehyde (Science; Munich, Germany) for 20 min at room temperature, subsequently washed twice with PBS without Ca2+ and Mg2+, blocked with 10% FCS serum (Vector; Loerrach, Germany) and 0.1% Triton X-100 (Sigma-Aldrich, Germany) in PBS and proceeded to immunocytochemistry with primary antibodies against OCT4, SOX-2, KLF-4, SSEA1, SSEA4, TRA-1-60 and TRA-1-81 from the hESC characterization kit (all 1:100, Millipore #SCR004), NANOG (1:100, Abcam #ab62734, Cambridge, UK), Smooth-Muscle-Actin (SMA) (1:100, Dako #M0851, Hamburg, Germany), Alpha-Fetoprotein (AFP) (1:100, Sigma-Aldrich #WH0000174M1), SOX17 (1:50, R&D #AF1924, Minneapolis, MN), PAX6 (1:300, Covance #PRB-278P, Münster, Germany), Nestin (1:200, Chemicon #MAB5326, Nürnberg, Germany), b-Tubulin III (1:1000, Sigma-Aldrich #T8660), Brachyury (T) (1:50, R&D #AF2085). Alexa-488-conjugated secondary antibodies were used (1:300, Invitrogen #A11001). Nuclei were counter-stained with DAPI (200 ng/ml, Invitrogen #H357) and visualized using the confocal microscope LSM510 (Carl Zeiss, Jena, Germany).