Fig 2: CT-179 and POx-Palbociclib dual regimen is more effective than either single-agent treatment.(A) Representative images of OLIG2/SOX10/DAPI stained MBs in G-Smo mice treated with CT-179 (100 mg/kg) or saline every three days.(B) Quantification of OLIG2+/SOX10- cells in MBs of G-Smo mice after 8 hours (left panel) and 5 days (right panel) of commencing treatment (means ± SD, n>3, **p < 0.01).(C) Cell-cycle occupation analysis of MBs from G-Smo mice treated with 80 mg/kg CT-179, 25 mg/kg POx-Palbociclib, or CT-179-POx-Palbo combination and harvested 6 hours after treatment (means ± SD, n=3 for each group).(D) Quantification of percentage of cells in G0 phase (left panel) G2/M phase (middle panel) and M phase defined by very high phosphorylated (p-RB++; right panel) in G-Smo mice after 6 hours of treatment (means ± SD, n=3, **p < 0.01).(E) Representative cleaved caspase-3 (cC3)/DAPI stained images from control, CT-179 (80 mg/kg), POx-Palbociclib (25 mg/kg), or combination (POx-Palbociclib + CT-179) treated tumors(F) Quantification of cC3 positive cells. Samples were harvested 6 hours after treatment.(G) Kaplan-Meier curves of G-Smo mice on the indicated treatment regimens (*p < 0.05, **p < 0.01, ***p < 0.001). The p values were determined by Log-Rank test.For panels B--D and F, the p values were determined by two-sided Student’s t-test.

Fig 3: MYC promotes UM formationA, Schematic diagram of Gnaq conditional activation, Bap1 conditional CRISPR knockout, and Myc conditional knock-in (GnaqCA; Bap1CKO; MycCKI) in the uvea by in situ delivery of pL-CLB. B, Kaplan-Meier overall survival curve of GnaqCA; Bap1CKO; MycCKI mice after pL-CLB administration. C, Representative in vivo bioluminescence imaging showing strong signal from a primary intraocular tumor. D and E, Representative images of H&E staining of intraocular tumors from GnaqCA; Bap1CKO; MycCKI mice. F, Representative fluorescent IHC showing SOX10 and RPE65 expression in a tumor from a GnaqCA; Bap1CKO; MycCKI mouse. Tumors are clonally positive for SOX10 and negative for RPE65 G, Representative image of Periodic acid–Schiff (PAS) staining of a tumor from a GnaqCA; Bap1CKO; MycCKI mouse. Tumors displayed minimal PAS positivity H, Representative images of IHC using a melanoma cocktail (Tyrosinase + Melan-A) to detect disseminated UM cells in the livers of GnaqCA; Bap1CKO; MycCKI mice. I, Schematic diagram of orthotopic and metastatic transplant of GnaqCA; Bap1CKO; MycCKI primary tumor cells in NSG mice. J and K, Representative images of H&E staining of orthotopic transplant of GnaqCA; Bap1CKO; MycCKI primary tumor cells (GMB12). L, Representative in vivo bioluminescence imaging showing signal from GMB12 cells 75 days after tail vein inoculation. M-O, Representative images of H&E staining of metastasis in lung (M), kidney (N), and liver (O). P, Representative IHC using a melanoma cocktail (Tyrosinase + Melan-A) on the liver of a mouse injected with GMB12 cells.

Fig 4: Generation of a genetically engineered mouse model of choroidal nevi driven by Gnaq mutation and Bap1 lossA, Schematic diagram of the Gnaq conditional activation (GnaqCA) allele. B, Mouse embryonic fibroblasts (MEFs) isolated from GnaqCA mice were infected by lentiviral Cre. PCR amplification and Sanger sequencing of Gnaq exon 5 from cDNA confirmed expression of the Q209L mutation. C, Western blot of Cre-infected GnaqCA MEFs revealed changes in Gαq downstream signaling but physiological GNAQ expression. D, Schematic diagram of Gnaq conditional activation (GnaqCA) in the uvea by in situ lentiviral delivery of Cre and Luciferase (pL-CL). E, Representative in vivo bioluminescence imaging of a GnaqCA mouse infected with pL-CL. F, Quantification of frequency of choroidal hyperplasia in GnaqCA mice at the indicated timepoints. G, Representative image of H&E staining showing localized choroidal hyperplasia in a GnaqCA mouse. H, Representative fluorescent IHC showing SOX10 and RPE65 expression in uveal tracts of wildtype and GnaqCA mice. I, Schematic diagram of Gnaq conditional activation and Bap1 conditional CRISPR knockout (GnaqCA; Bap1CKO) in the uvea by in situ lentiviral delivery of Cre, Luciferase, and sgBap1 (pL-CLB). J, Quantification of choroidal hyperplasia and tumors in GnaqCA; Bap1CKO mice at 7 months. K and L, Representative images of H&E staining of choroidal hyperplasia (K) and micro-tumor (L) in GnaqCA; Bap1CKO mice. M, Representative fluorescent IHC showing SOX10 and RPE65 expression in the uveal tract of a GnaqCA; Bap1CKO mouse.

Fig 5: Lacrimal gland hyperplasia in PLCγ1 conditional knockout mouse(A) Co-expression of the GFP reporter with the Le-Cre transgene illustrates the extension of the lacrimal gland bud during embryonic development and its subsequent branching into a glandular structure post-birth. The deletion of PLCγ1 resulted in extra buds from E15.5 to E16.5 (arrowheads), ectopic branches at P1 (arrow), and enlarged lacrimal glands at P30.(B) Quantification of branch number from E14.5 to E16.5. Student’s t test, n = 7 for control, n = 14 for mutant, *p < 0.0001.(C) Quantification of lacrimal gland size normalized against control at P30. Student’s t test, n = 6, p < 0.0001. Scale bars: 250 μm.(D) Immunostaining revealed the absence of PLCγ1 in the lacrimal gland bud at E14.5, while the expression level of progenitor cell marker Sox9 remained unaffected. In contrast, Sox10 expression expanded from the end bud to the stalk region, and the proliferation marker pHH3 was increased throughout the lacrimal gland.(E) Quantification of Sox10+ cells. Student’s t test, n = 5, *p < 0.01.(F) Quantification of pHH3+ cells. Student’s t test, n = 3, *p < 0.02.(G) At P1, PLCγ1-deficient glands exhibited a rise in Sox10+ and Ki67+ cells, contributing to an increased number of Mist1-expressing acinar cells. By P30, these glands were densely packed with diminished interstitial space.(H) Quantification of Ki67-expressing cells. Student’s t test, n = 3, p < 0.001.(I) Quantification of Mist1-expressing cells. Student’s t test, n = 6 for control, n = 3 for mutant, p < 0.0001. Scale bars: 50 μm.Data are represented as mean ± SEM.