D4476

SR3029, D4476, MG149, NU9056, MG132 and cycloheximide (CHX) were purchased from MedChemExpress (MCE, Monmouth Junction, NJ, USA). The following primary antibodies were used: anti-CK1δ, anti-β-catenin, anti-p300, anti-acetyl-lysine, IgG (Santa Cruz Biotechnology, Heidelberg, Germany), anti-CBP, anti-acetyl-β-catenin (K49), anti-phospho-β-catenin (S45), anti-Flag, anti-V5, anti-CK1ϵ, anti-GFP, anti-mouse IgG (Cell Signaling Technology, Danvers, MA), anti-Tip60 (Abcam, Cambridge, MA, USA), and anti-GAPDH antibody (Proteintech, Chicago, IL, USA). The Tip60-Flag, PCAF-Flag, p300-Flag and pEGFP-N1 plasmids were purchased from Vigenebio (Weizhen, Shandong, China). The SuperTopFlash reporter plasmid was provided by Karl Willert (University of California at San Diego, La Jolla, CA, USA). The expression plasmids encoding β-catenin, pCMXβgal (β galactosidase, β-gal), CK1α-V5, CK1δ-V5, CK1ϵ-V5, CK1α-Flag, CK1δ-Flag, CK1ϵ-Flag, CK1γ-Flag have been described previously (35 (link), 36 (link)). For the construction of GFP-tagged CK1δ, the cDNAs encoding human CK1δ was amplified by PCR and subcloned into the BamHI/EcoRI site of pEGFP-N1 vector using ClonExpress Ultra One Step Cloning Kit (Vazyme, Nanjing, China). The primer sequences used are as follows: CK1δ-GFP-sense, 5’-CTCGAGCTCAAGCTTCATGGAGCTGAGAGTCGGGAAC-3’; CK1δ-GFP-antisense, 5’-CTCACCATAAGGTGGCGACCGGTGTAGGTGCGTCGTG.

A549 and NCI-H23 lung cancer cell lines were obtained from Dr. Massimo Broggini (IRCCS—Istituto di Ricerche Farmacologiche Mario Negri, Milan, Italy). The HEK293T cell line was purchased from ATCC (LGC Standards, Sesto S. Giovanni, Italy). All cell lines were sub-cultured in RPMI-1640 medium (Thermo Fisher) supplemented with 10% fetal bovine serum (FBS, Euroclone, Milan, Italy) and antibiotics at 37 °C and 5% CO₂. All cell lines are authenticated through SNP profiling by Multiplexion Gmbh (Heidelberg, Germany) and are routinely checked for mycoplasma contamination. Cell lines were treated for 72 h with a range of concentrations of D4476 (MedChem Express, Monmouth Junction, NJ, USA), daminozide (MedChem Express, Monmouth Junction, NJ, USA), or LY255283 (Santa Cruz Biotechnology, Dallas, TX, USA), then dissolved in DMSO and further diluted in complete medium. DMSO alone was used as treatment control. Cell growth was monitored using the Incucyte^® S3 live cell imaging system (Essen Biosciences Inc, Ann Arbor, MI, USA) and EC50 curves were generated using Incucyte^® software (2020B version, Essen Biosciences Inc, Ann Arbor, MI, USA).

GFP-AZI2 -4OHT cells were seeded in 24-well plates and left overnight before treatment with respective inhibitors. Upon treatment, images of cells were acquired every 2 h with an Incucyte live-cell imaging system (Essen Bioscience) to monitor GFP-AZI2 puncta formation. Inhibitor concentrations used in this screen were; SB203580, 10 µM (MedChemExpress, HY-10256); Tozasertib, 5 µM (MedChemExpress, HY-10161); Chloroquine, 100 µM (Sigma Aldrich, C6628); GDC0994, 5 µM (Cayman Chemical, 21107); Prexasertib, 5 µM (MedChemExpress, HY-18174); LTURM34, 10 µM (MedChemExpress, HY-101667); GSK650394, 5 uM (MedChemExpress, HY-15192); Amlexanox, 100 µM (MedChemExpress, HY-B0713); U0126, 10 µM (Cayman Chemical, 70970); IRAK 1–4 Inhibitor I, 10 µM (MedChemExpress, HY-13329); SP600125, 25 µM (MedChemExpress, HY-12041); SBI0206965, 10 µM (Cayman Chemical, 18477); BMS345541, 10 µM (MedChemExpress, HY-10519); Triciribine, 10 µM (MedChemExpress, HY-15457); Silmitasertib, 10 µM (MedChemExpress, HY-50855); PP242, 5 µM (MedChemExpress, HY-10474); Lys05, 20 µM (provided by Dr. Ravi Amaravadi); Palbociclib, 5 µM (MedChemExpress, HY-50767); and D4476, 50 µM (MedChemExpress, HY-10324). The puncta/image at 24 h were quantified and plotted.