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. 2021 Feb 18;81(4):767–783.e11. doi: 10.1016/j.molcel.2020.12.006

Figure 1.

Figure 1

Loss of Alc1 does not affect lifespan and reduces DEN-induced tumor occurrence

(A) Top: Schematic representation of mouse Alc1 genomic locus. The gene-trap vector rsFROSAbgeo0s is inserted at position 4827 in intron 1. The whole genomic locus is 49.461 kb, and introns (lines) and exons (bars) are approximately to scale. Gray lines represent primers used for genotype. Bottom: ALC1 protein organization. Alc1 mutant protein is truncated at the 45th amino acid and fused to the β-Geo cassette of the gene-trap vector.

(B) Weight analysis of Alc1+/+ and Alc1−/− mice. Error bars are not shown to render the graph readable; data are from males and females with at least five mice measured at each time point.

(C) Tumor-free survival of Alc1 mice. Significance: Mantel-Cox test, p = 0.4. n = 30 Alc1+/+ and n = 30 Alc1−/−. Mice culled due to nonspecific phenotypes (e.g., dermatitis, overgrown teeth, and fits) were excluded from this study. Right: Frequency of Alc1 mice that develop tumors. Note that there is no difference between both groups.

(D–F) Alc1−/− mice show reduction in both spontaneous epithelial and mesenchymal and DEN-induced tumor formation. (D) Epithelial and mesenchymal tumor-free survival of Alc1 mice. Significance: Mantel-Cox test, p = 0.1. n = 30 Alc1+/+ and n = 30 Alc1−/−. Mice culled due to nonspecific phenotypes (e.g., dermatitis, overgrown teeth, and fits) were excluded from this study. Right: Frequency of Alc1 mice that develop epithelial or mesenchymal tumors. Note that there is a tendency for Alc1−/− mice to develop less epithelial and mesenchymal tumors. Significance: Fisher’s exact test, p = 0.2. (E) Left: Representative images of epithelial tumors. Note the presence a stomach adenoma with peculiar hyaline pink cells in the Alc1+/+ mouse. Scale bars represent 100 μm. Right: Representative images of mesenchymal tumors. Note the presence an hemangiosarcoma in the spleen of Alc1+/+ mouse. Scale bars represent 100 μm. (F) Left: Pictures of liver from 36-week-old Alc1+/+ and Alc1−/− male mice intraperitoneally injected with DEN (25 mg/kg body) at 2 weeks of age and fed with high-fat diet. n = 6. Right: Tumor size measurement in mm. Each tumor has been measured with a caliper. Note the smaller-sized tumors in the Alc1−/− group. Significance: t test, p < 0.0001.

(G and H) Alc1−/− MEFs are sensitive to PARPi. (G) Reduced survival of Alc1−/− MEFs after treatment with Olaparib. Data are mean ± SEM normalized to untreated cells (n = 3 biologically independent experiments). (H) Growth curves in Alc1+/+ and Alc1−/− MEFs in non-treated controls and with indicated Olaparib doses. Data are mean ± SEM (n = 3 biologically independent experiments).

(I) Reduced survival of Alc1−/− MEFs after treatment with MMS. Data are mean ± SEM normalized to untreated cells (n = 3 biologically independent experiments).ns, p > 0.05; ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001.