Figure S5.
c-MYC inhibition restores R-spondin levels. (A) Scheme depicting chemical inhibition of c-MYC by JQ1 in URI lox mice. (B) Representative pictures of H&E staining and IHC of URI in intestinal sections from JQ1-treated URI(+/+)Int, vehicle-treated URI(Δ/Δ)Int and JQ1-treated URI(Δ/Δ)Int mice at indicated time points of tamoxifen treatment. (C) Kaplan–Meier curve of JQ1-treated URI(+/+)Int (black solid line; n = 4), vehicle (5% DMSO and 5% glucose in water)-treated URI(Δ/Δ)Int (red solid line; n = 6) and JQ1-treated URI(Δ/Δ)Int mice (yellow solid line; n = 7). (D) Representative pictures of IHC of c-MYC, γH2AX, phospho-RPA, p53, cleaved caspase 3, caspase 1, and chromogranin A and Alcian blue/PAS staining from vehicle-treated URI(Δ/Δ)Int and JQ1-treated URI(Δ/Δ)Int mice following 6 and 9 d of tamoxifen treatment, respectively. Graph represents number of c-MYC positive cells per crypt. Black arrows represent caspase 1 positive foci. Graphs represent number of positive cells per crypt (n = 3, 4, 5, 6). (E) qRT-PCR of Rspo1 and Rspo3 mRNA levels of vehicle-treated URI(Δ/Δ)Int and JQ1-treated URI(Δ/Δ)Int mice (n = 3). (F) qRT-PCR of Rspo1 mRNA levels of URI(+/+)Int, URI(Δ/Δ)Int, and URI(Δ/Δ)Int; APC(Δ/Δ) mice (n = 4, 4, 5). (G) Scheme depicting c-MYC genetic ablation in URI lox mice. (H) Representative IHC of cMYC in intestinal sections from URI(Δ/Δ)Int; c-MYC(+/+)Int and URI(Δ/Δ)Int; c-MYC(Δ/Δ)Int mice after 6 d of tamoxifen treatment. (I) Representative pictures of IHC of URI in intestinal sections from URI(+/+)Int; c-MYC(Δ/Δ)Int, URI(Δ/Δ)Int; c-MYC(+/+)Int, and URI(Δ/Δ)Int; c-MYC(Δ/Δ)Int mice at indicated time points of tamoxifen treatment. Data represent mean ± SEM; *, P < 0.05; **, P < 0.01; ***, P < 0.001; Mantel–Cox, Student’s t test and one-way ANOVA. Scale bars represent 20 μm in D and H; 50 μm in D (Alcian Blue/PAS and chromogranin A panels); and 100 μm in B and I. At least 50 crypts and 100 villi were quantified per mouse. H&E and IHC are representative of at least three independent mice.

c-MYC inhibition restores R-spondin levels. (A) Scheme depicting chemical inhibition of c-MYC by JQ1 in URI lox mice. (B) Representative pictures of H&E staining and IHC of URI in intestinal sections from JQ1-treated URI(+/+)Int, vehicle-treated URI(Δ/Δ)Int and JQ1-treated URI(Δ/Δ)Int mice at indicated time points of tamoxifen treatment. (C) Kaplan–Meier curve of JQ1-treated URI(+/+)Int (black solid line; n = 4), vehicle (5% DMSO and 5% glucose in water)-treated URI(Δ/Δ)Int (red solid line; n = 6) and JQ1-treated URI(Δ/Δ)Int mice (yellow solid line; n = 7). (D) Representative pictures of IHC of c-MYC, γH2AX, phospho-RPA, p53, cleaved caspase 3, caspase 1, and chromogranin A and Alcian blue/PAS staining from vehicle-treated URI(Δ/Δ)Int and JQ1-treated URI(Δ/Δ)Int mice following 6 and 9 d of tamoxifen treatment, respectively. Graph represents number of c-MYC positive cells per crypt. Black arrows represent caspase 1 positive foci. Graphs represent number of positive cells per crypt (n = 3, 4, 5, 6). (E) qRT-PCR of Rspo1 and Rspo3 mRNA levels of vehicle-treated URI(Δ/Δ)Int and JQ1-treated URI(Δ/Δ)Int mice (n = 3). (F) qRT-PCR of Rspo1 mRNA levels of URI(+/+)Int, URI(Δ/Δ)Int, and URI(Δ/Δ)Int; APC(Δ/Δ) mice (n = 4, 4, 5). (G) Scheme depicting c-MYC genetic ablation in URI lox mice. (H) Representative IHC of cMYC in intestinal sections from URI(Δ/Δ)Int; c-MYC(+/+)Int and URI(Δ/Δ)Int; c-MYC(Δ/Δ)Int mice after 6 d of tamoxifen treatment. (I) Representative pictures of IHC of URI in intestinal sections from URI(+/+)Int; c-MYC(Δ/Δ)Int, URI(Δ/Δ)Int; c-MYC(+/+)Int, and URI(Δ/Δ)Int; c-MYC(Δ/Δ)Int mice at indicated time points of tamoxifen treatment. Data represent mean ± SEM; *, P < 0.05; **, P < 0.01; ***, P < 0.001; Mantel–Cox, Student’s t test and one-way ANOVA. Scale bars represent 20 μm in D and H; 50 μm in D (Alcian Blue/PAS and chromogranin A panels); and 100 μm in B and I. At least 50 crypts and 100 villi were quantified per mouse. H&E and IHC are representative of at least three independent mice.

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