Icg 001

GFP⁺ lentivirus-transduced erythroblasts (CD71⁺/Ter119^-) were seeded at a density of 1 × 10⁶ cells/mL in StemPro-34 SFM (ThermoFisher) supplemented with l-glutamine (1%; ThermoFisher), Epo (Peprotech; 10 U/mL) and transferrin (Sigma-Aldrich; 1 mg/mL) as previously published^{36 (link)}. The GFP⁺ cells were cultured for 48 h in the presence and absence of ICG001 (Tocris) and JW74 (Tocris). Half media exchange was performed after 24 h, and after 48 h the cells were collected, stained with CD71 and Ter119 antibodies and analyzed using flow cytometry.
For experiments involving FL cells, FACS sorted CD71⁺Ter119^- FL cells were seeded at a density of 0.5 × 10⁶ cells/mL StemPro-34 SFM (ThermoFisher) supplemented with l-glutamine (1%; ThermoFisher), Epo (Peprotech; 10 U/mL) and transferrin (Sigma-Aldrich; 1 mg/mL). The FL cells were cultured for 48 h in the presence and absence of ICG001 (Tocris) and JW74 (Tocris). Half media exchange was performed after 24 h, and after 48 h the cells were collected, stained with CD71 and Ter119 antibodies and analyzed using flow cytometry.

The experimental protocol was approved by the Ethics Committee for the Use of Experimental Animals in Zhejiang University (No. 2016‐279) and was carried out in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 80‐23). Two‐month‐old male C57BL/6 mice were provided by the Experimental Animal Center in Zhejiang Academy of Medical Sciences (Hangzhou, Zhejiang, China). Mice were maintained on standard rodent chow and had free access to food and water under a 12/12 h light/dark cycle, and BrdU was from Sigma (St. Louis, MO, USA). PDF with 4.25% glucose was from Baxter (Chicago, IL, USA). ICG‐001 was from Tocris Bioscience (Ellisville, MO, USA). Antibodies against β‐catenin, E‐cadherin, and Vimentin were from Cell Signaling Technology (Danvers, MA, USA). Antibodies against Snail and GSK‐3β were from Abcam (Cambridge, MA, USA).

PKF115-584-mediated inhibition of β-catenin activity in pmATII cells was performed by treatment with a concentration of 1 µM beginning at day 1 of culture until day 3 and day 5, respectively. Control cells were treated with the corresponding concentration of DMSO. Treatment and control media were refreshed at day 3. ICG-001 treatment (7.5 µM) was applied in the same manner. CT99021 treatment (2 µM) of cells was performed from day 1 to day 3. DMSO-treated cells served as control. The pharmacological inhibitors were purchased from the following companies: PKF115-584 (Santa Cruz), ICG-001 (Biomol) CT99021 (Tocris). siRNA-mediated downregulation of Ctnnb1, Eno1 or Pdia3 (ERp57) was performed using an siRNA pool of three target-specific siRNAs (Ctnnb1 siRNA, α-Enolase siRNA, ERp57 siRNA, Santa Cruz Biotechnology, Dallas, TX). Cells were transfected at day 2 after isolation using Lipofectamine RNAiMAX Transfection Reagent (Life Technologies) according to the manufacturer's instructions. Cells transfected with a scrambled control siRNA (siScr) (Santa Cruz Biotechnology, Dallas, TX) served as control. Non-transfected cells served as additional control. Cells were analyzed at day 5. siRNA efficiency was confirmed on mRNA level (data not shown) and protein level (Figs 4C and 6A,B).