Fig 2: AP2M1 expression following apoptosis induction.a HCT116 and HCT116 p21-/- cells have been treated with sn38 for 72 h (5 ng/ml) and apoptosis was evaluated by FACS analysis and the detection of subG1 cells (n = 3 ± s.d.). b Phospho-AP2M1 and p21waf1 expressions have been analyzed by western blot in the indicated cells (n = 3). c, d LS174T or HCT116 cells were treated with sn38 in the presence or absence of Akti 1/2 for 72 h (10 µM) and phospho-AP2M1 and p21waf1 expressions were analyzed as described above (n = 3)

Fig 3: AP2M1 regulates CIS escape and is involved in the transmission of soluble signals from senescent cells.a LS174T cells have been transfected with the corresponding siRNAs and protein expression was analyzed by western blot (n = 3). b Following treatment and siRNA transfection, emergence was evaluated after 10 days. Representative images are shown on the left part of the figure and the quantification of emergent clones is presented on the right part (n = 6; **p < 0.01). c Conditioned media (CM) from parental or senescent cells were collected after 24 h of serum starvation. Migration assays were performed using Boyden inserts. RPMI or conditioned media supplemented with 3% FBS were deposited at the bottom of the well, parental LS174T cells at the top. After 72 h, migrating cells were stained with crystal violet (n = 3). d RPMI or conditioned media collected as above supplemented with 3% FBS were deposited at the bottom of the the well. Parental LS174T cells or cells transfected with the indicated siRNAs were deposited at the top to evaluate the sensitivity of cells expressing or not AP2M1 to soluble signals generated from senescent cells. After 72 h, migrating cells were stained with crystal violet (n = 3)

Fig 4: SGC-AAK1-1 Is a Potent and Specific Inhibitor of AAK1 and BMP2K(A) Chemical structures of the AAK1/BMP2K chemical probe SGC-AAK-1, the related AAK1/BMP2K inhibitor 25A, and the negative control compound 34A that has a similar chemical structure but does not inhibit AAK1 or BMP2K.(B) SGC-AAK-1 selectively bound AAK1 and BMP2K with a more than 30-fold difference in Ki in a TR-FRET binding displacement assay over the related kinases GAK and STK16. Sixteen-point dose-response curves were measured in duplicate.(C) SGC-AAK1-1 has good selectivity over the human kinome. SGC-AAK1-1 was used at 1 µM concentration and tested against over 400 wild-type human kinases. KD determination was completed for all kinases that exhibited >85% inhibition and/or were observed as common off-targets for this structural class. Kinases for which SGC-AAK1-1 has a KD < 100 nM are marked with a large red circle and 100 nM < KD < 1.0 µM with a smaller red circle.(D) ITC confirmed a KD of 120 nM for binding of SGC-AAK1-1 to AAK1.(E) A co-crystal structure of BMP2K (2.41 Å) bound to 25A revealed the binding mode of the inhibitor in the ATP site. This structure revealed five key hydrogen bonds and revealed the orientation of the central hydrophobic portion of the inhibitor with respect to the binding pocket.(F and G) NanoBRET cellular target engagement assays in HEK293T cells showed that 25A (F) and SGC-AAK1-1 (G) both entered cells and directly bound to AAK1 and displaced a fluorescent tracer molecule from the ATP site. Cells were treated with serially diluted inhibitors in the presence of four different concentrations of a NanoBRET tracer molecule, and IC50 values were calculated from a linear interpolation of the IC50 values at each tracer concentration to obtain the predicted IC50 in the absence of tracer. Measurements were made with three technical replicates.(H) Binding of SGC-AAK1-1 to BMP2K in cells was weaker than to AAK1 with IC50 > 1 µM. Displacement of the same fluorescent tracer molecule required increased concentrations of SGC-AAK1-1, revealing an elevated IC50 value for binding to BMP2K versus AAK1 in the cellular target engagement assay.(I) SGC-AAK1-1 inhibited the phosphorylation of the AP2M1 subunit at Thr156 in HEK293T cells in a concentration-dependent manner or with 10 µM 34A. HEK293T cells were treated with inhibitors for 2 hr before western blot analysis.Error bars represent SE. See also Figure S1 and Tables S2, S3, S4, and S5.

Fig 5: WNT Treatment Induces AAK1-, PIP2-Dependent Phosphorylation of AP2M1(A) Western blot analysis of HT1080 cells treated with WNT3A CM for indicated times.(B) Western blot analysis of HT1080 cells transfected with either AAK1 or control siRNA for 72 hr. Cells were then treated with rhWNT3A (200 ng/mL) for 15 min, then the medium was changed for fresh, complete DMEM, and cells were incubated for 9 hr.(C) Western blot analysis of pAP2M1 levels in HEK293T cells treated with either rhWNT3A (3A) or rhWNT5A (5A) (200 ng/mL) for 9 hr. The box-and-whisker plot represents pAP2M1/AP2M1 expression from four biological replicates quantified by LiCOR software.(D) Western blot analysis of HT1080 cells treated with either CHIR99021 compound (1 µM) or rhWNT3A (200 ng/mL) for the indicated time.(E) HEK293T cells were transfected with FLAG-dnTCF7L2 for 24 hr. Cells were then treated with WNT3A for 15 min, then the medium was changed for fresh, complete DMEM, incubated for 9 hr, and analyzed using western blot.(F) Western blot analysis of HEK293T WT, DVL TKO clone #4, DVL TKO clone #6, and LRP5/6 DKO cells treated with WNT3A CM for 9 hr.(G) Western blot analysis of HT1080 cells treated with WNT3A CM for 15 mins, then the medium was changed for complete DMEM containing either carbachol (50 µM) or neomycin (100 µM). Cells were incubated for 9 hr prior to cell harvest.(H) Western blot analysis of HT1080 cells transfected with siRNA against PI4K2a for 72 hr and pulsed with WNT3A CM for 15 mins and then incubated for 9 hr.All panels are representative of biological triplicates, unless otherwise noted. For complete statistics, see STAR Methods.See also Figure S4.