Cki 7

HEK293 cells were grown in DMEM medium supplemented 10% FBS (HyClone), 2 mM L-glutamin at 37 °C, 5% CO2. The cells were treated by CHX 20mkM or 6 and 12 uM (as indicated) of CKI-7 (Sigma). HEK293 cells were lysed in Lysis Buffer: 10 mM HEPES (pH 7.9) containing 5 mM MgCl, 0.5% Nonidet P-40, 0.45 M NaCl, 1 mM DTT, a protease inhibitor cocktail (PIC) (Roche) and a 1% Phosphatase inhibitor cocktail 3 (PhIC) (Sigma-Aldrich). The lysate was centrifuged at 10 000 rpm, 4 °C, for 10 min, and the supernatant was diluted 4-fold with the same buffer but without NaCl. The extract was treated with DNAse I (USB, 0.6 units/mL) and RNAse (Stratagene, 10 units/mL).
For cellular fractionation cells were lysed in FLB buffer (40 mM Tris-HCl, pH = 7,8; 100 mM NaCl; 2,5 mM MgCl2, 1 mM DTT; PIC; PhIC) on ice, grinded in Loose Dounce, centrifuged for 1 minute and supernatant was employed as the cytoplasmic fraction. The pellet was resuspended in Lysis Buffer, grinded in Thight Dounce, incubated 10 minutes on ice, centrifuged as mentioned above and diluted the same way.
The probes were equalized using Qubit Protein Assay Kit (ThermoFisher Scientific), mixed with 4X LB (200 mM Tris-HCl, pH = 6.8; 4% SDS; 40% Glycerol; ~0,01% Bromphenol blue; 100 mM DTT), and boiled for 10′.
Quantification of the Western blots was done using ImageJ gel analysis software (NIH, USA)⁴² .

In our study, P0–P3 hAESCs were chosen for investigation. For PR–like cell differentiation, hAESCs were cultured in knockout medium comprising DMEM/F12, 15% (vol/vol) KnockOut serum, 2 mM glutamine, 1 × nonessential amino acids, 1 × antibiotic–antimycotic, 1% B27 supplement (Thermo Scientific), and 1% N2 supplement (Thermo Scientific) in plates pretreated with fibronectin. Then, 5 μM SB–431542 (Sigma–Aldrich, Saint Louise, MO, USA, Cat# C0742), 5 μM CKI–7 (Sigma–Aldrich, Cat# S4317), and 10 ng mL⁻¹ human noggin (Peprotech, Cat# 120–10C) were added to the medium for approximately 10 days. Then, 1 μM retionic acid (Sigma–Aldrich, Cat# R2625), 100 μM taurine (Sigma–Aldrich, Cat# T8691), and 10 ng mL⁻¹ human noggin (Peprotech) were supplemented during the second week. The medium was replaced every other day, and after 12 days of culture, the cells were passaged to a fibronectin–coated plate at a 1:3 ratio.

hESC colony cultures were incubated with the Rho Kinase inhibitor Y-27632 (Rock Inhibitor, 10 μM: Sigma-Aldrich, St Louis, MO, USA) for 1 h and dissociated to single cells using TrypLE™ Express. Cell aggregates (1000–1500 cells) were formed using the AggreWell™400 system (StemCell Technologies) and cultured in Basal Differentiation Media (BDM) with Rock Inhibitor (Table S1). For differentiation, aggregates were cultured in BDM supplemented with combinations of SB43218 (SB: 10 μM), CKI-7 (CKI: 5 μM) and nicotinamide (NIC: 10 mM) (all Sigma-Aldrich). After 6 days, embryoid bodies (EBs) were plated on GFR-MG-coated plates in BDM.
At day 8, expanding EBs were dissociated with collagenase IV (1 mg/mL) and re-plated as clumps of 200 to 500 cells on GFR-MG-coated 24-well dishes. Cells were then cultured in BDM without factors and refed every 2 to 3 days until day 38.

The study was approved by the ethical committees of the Institute of Biomedical Research and Innovation Hospital and the RIKEN Center for Developmental Biology, Japan. Human iPSC-lines 201B7 (HPS4290) and 253G1 (HPS0002) (Nakagawa et al., 2008 (link)) were provided by the RIKEN BRC through the National BioResource Project of the MEXT/AMED, Japan. iPSCs were cultured and differentiated into RPE cells as described previously (Kamao et al., 2014 (link)). Briefly, to differentiate human iPSCs into RPE cells, human iPSCs were cultured on gelatin-coated dishes in differentiation medium (GMEM (Sigma-Aldrich, St. Louis, MO) supplemented with 1 mM sodium pyruvate, 0.1 mM non-essential amino acids (Sigma-Aldrich), and 0.1 mM 2-mercaptoethanol (Sigma-Aldrich)) with 20% KnockOut Serum Replacement (KSR; Invitrogen, Waltham, MA) for four days, 15% KSR for six days, and 10% KSR for 20 days. Y-27632 (10 μM; FUJIFILM Wako, Osaka, Japan), SB431542 (5 μM; Sigma-Aldrich), and CKI7 (3 μM; Sigma-Aldrich) were added for the initial 18 days. After the emergence of pigmented cells, the medium was switched to SFRM (DMEM/F12 [7:3] supplemented with B27 (Invitrogen), 2 mM L-glutamine).
Lonza-RPE cells (line #476621; Lonza, Basel, Switzerland) were maintained in SFRM as well.
Human dermal fibroblasts were obtained from a healthy donor and cultured as described previously (Sugita et al., 2015 (link)).

For in vitro kinase assay linker domain (238–361 aa) and A-Mutant forms were clone in pET22b vector and then recombinant 6His-Linker Domain proteins expressed in E. coli and purified at Ni Sepharose (GE Healthcare). For the in vitro kinase experiments, 1 ug 6His-Linker Domain or the same ammount of mutants forms were incubated with 100 ul of HEK293 extracts or extracts with inhibitors of 12 uM CK-1 (CKI-7, Sigma-Aldrich) or GSK-3ß (CHIR 99021, Sigma-Aldrich) in the presence of γ-³² [P]-labeled ATP at 37 °C. Than the samples were processed for autoradiography or western blot as specified.