Supplemental Figure 1: Uncut gels

A. Media Lysate B.

Unt 0 0.01 0.1 1 C59 (nM) 10 Unt 0 0.01 0.1 1 10 Wnt8a-V5+C59 (10nM) Wnt8a-V5 w/o Wnt (EV ) Wnt3a-V5 Wnt3a-V5+C59 (10nM )

250 w/o Wnt (EV ) 150

100 exposure short 75 50 Wnt-V5 37 IP

Wntless

Wnt-V5 250 150 100 75 long exposure 50 Lysate 37 Wntless

25 20

Streptavidin-800 V5, Dylight-680 C. V5 IP + click lysate lysate V5 IP + click w/o + lysate lysate click click V5 IP no click w/o + V5 IP no click C59 IWP DMSO

No Alk click click C59 IWP DMSO No Alk

HT1080 75 HT1080 50 37 25

75 HeLa 50 HeLa 37 25

Overlay

lysate lysate V5 IP + click D. w/o + EV WNT3A WNT4 WNT5A WNT11 WNT16 click click V5 IP no click C59 IWP DMSO No Alk 250 150 75 HT1080 50 100 37 75 Dvl2-p 25 Dvl2

50 75 HeLa 50 37 25 C59: - - + - + - + - + - + Supplemental Figure 1 (A) Treatment with C-59 blocks the secretion of WNT3A into the media: STF cells stably transfected with Wnt3a were treated with DMSO or indicated concentrations of C-59. Culture media was collected and the cell lysates were prepared after 24 h. Western blots were performed to detect WNT3A. (B) C59 blocks Wnt-WLS interaction: HeLa cells were transfected with either WNT3A-V5 or WNT8A-V5 plasmids, then treated with DMSO or C59 at 10 nM. WLS was immunoprecipitated and precipitates were probed for WLS and V5 to determine the interaction. Lysates were also probed for total V5-tagged WNTs. (C) C59 blocks palmitoylation of Wnts: Alkyne palmitic acid (Alk-C16) was fed to HeLa or HT1080 cells transfected with WNT3A-V5 were treated with DMSO, C59 (100 nM), or IWP1 (1 µM). Lysates were prepared and Wnts were immunoprecipitated with antibody to V5. Click chemistry was performed to attach azido-biotin to alkyne-palmitate groups. Finally, samples were separated by SDS-PAGE and probed for biotin and WNT3A-V5. (D) Dvl2 mobility shift was measured in HT1080 cells transfected with Wnts indicated, in the presence or absence of 10 nM C59.

Supplemental Figure 2 A. B. p = 0.0525 Vehicle 1.5 * 3000 ** C 59- t ) p g i r m c (

s t 1.0 n h 2000 a g i r e T

W e

r v i o 1000 t 0.5 a m l u e T R 0 0.0 Vehicle C59 Axin2 c-Myc CyclinD2 Tcf7 Target

C. 120 ght

e i 100 W

od y

B 80 C59 (n=5) a l i t Vehicle (n=6) n i I 60 %

1 3 5 7 9 11 13 15 17 19 21 Days of Treatment

Supplemental Figure 2: (A) C59 significantly decreased tumor weight: Tumor weights at sacrifice from the primary MMTV-WNT1 tumors are shown. (B) C59 hits the β-catenin target genes: Total RNA was isolated from tumors, and mRNA for Axin2, CyclinD2, C-Myc and Tcf7 were measured by qRT-PCR. Expression was normalized to actin. *p-value = 0.001, two-tailed t-test. (C) C59 is not toxic at effective doses: Total body weights are plotted for individual mice. Vehicle C59

Supplemental Figure 3: C59 decreases cytoplasmic and nuclear β-catenin in tumors: β-catenin staining of MMTV-WNT1 tumor sections. Scale bars = 50 µm.

See Figure 4D Legend for Experiment Description Supplemental Figure 4 Cerebellum Cerebrum Lungs

Fat Pad Heart Kidney

Spleen Thymus Liver

Bladder Uterus Ovaries

Skin Small Intestine Supplemental Figure 4: C59 is non- toxic: Representative images from hematoxylin and eosin stained tissues of a MMTV-WNT1 mouse treated with 5mg/kg C59 for 21 days.