Figure 2. Suppression of stemness properties of 4T1 cells by CLOCK.

(A) CLOCK protein levels in mock-transduced and Clock-expressing lentivirus-infected 4T1 cells. Values show mean with SD (n = 3). **p<0.01; significant difference from mock-transduced 4T1 cells (t₄ = 15.736, Student’s t-test). (B) The flow cytometric analysis of ALDEFLUOR assay of CLOCK-expressing 4T1 cells. The population of ALDH-positive cells was defined based on each DEAB group, whose means of fluorescence intensity were set almost the same. The right panel shows the difference in the ratio of ALDH positive- to negative-cell populations between mock-transduced and CLOCK-expressing 4T1 cells. Values show the mean with SD (n = 6). **p<0.01; significant difference from mock-transduced 4T1 cells (t₁₀ = 22.455, Student’s t-test). (C) Difference in the mRNA levels of ALDH isoforms between mock-transduced and CLOCK-expressing 4T1 cells. Values show the mean with SD (n = 3). (D) The mRNA levels of stemness-related genes in ALDH-positive mock-transduced and CLOCK-expressing 4T1 cells. Data were normalized by the18s rRNA levels. Values show the mean with SD (n = 3). The values of the mock-transduced group are set at 1.0. **p<0.01: significant difference from mock-transduced 4T1 cells (t₄ = 9.261 for Klf4; t₄ = 8.001 for Nanog; t₄ = 32.576 for Myc; Student’s t-test). (E) Difference in growth ability between mock-transduced and CLOCK-expressing 4T1 cells. Values show the mean with SD (n = 5–6). Cell viability of seeding day (day 0) are set at 1.0. **p<0.01; significant difference between from mock-transfected 4T1 cells at corresponding time points. (F_{7, 38} = 425.953, two-way ANOVA with the Tukey–Kramer test). (F) Difference in the spheroid formation ability between mock-transduced and CLOCK-expressing 4T1 cells. The left panel shows a representative photograph of the Hoechst-stained spheroids formed by mock-transduced or CLOCK-expressing 4T1 cells. The right panel shows the number of spheroid and the parcellation of the diameter. Values show the mean with SD (n = 3). **p<0.01; significant difference from mock-transduced 4T1 cells (t₄ = 27.067, Student’s t-test, for number of spheroids).

Figure 2—figure supplement 1. CLOCK regulates the expression of Aldh3a1 through mediation of C/EBPα.

(A) Procedure of selecting transcriptional factors that regulate Aldh3a1 expression in 4T1 cells. Genomic DNA binding prediction database (JASPAR) and ChIP Atlas database were applied to identify factor that mediates CLOCK-regulated Aldh3a1 expression. (B) The expression levels of C/EBPα mRNA in enhanced CLOCK-expressing 4T1 cells. Values are the mean with SD (n = 3). **p<0.01; significant difference between two groups (t₄ = 5.703, Student’s t-test). (C) The alignment of promoter sequences of Aldh3a1 genes in mouse (Mus musculus), rat (Ratta norvegicus), and human (Homo sapiens). (D) Repression of Aldh3a1 transcriptional activity by C/EBPα. Luciferase reporter vectors containing −2000 bp upstream region of the mouse Aldh3a1 gene (Aldh3a1 [−2000]::Luc) were constructed. 4T1 cells were transfected with Aldh3a1 [−2000]::Luc and C/EBPα-expressing vectors. Values are the mean with SD (n = 3). The values of pcDNA group are set at 1.0. **p<0.01; significant difference between two groups (t₄ = 19.351, Student’s t-test). (E) Repression of Aldh3a1 expression by C/EBPα. 4T1 cells were transfected with C/EBPα-expressing vectors. Values are the mean with SD (n = 3). The values of pcDNA group are set at 1.0. **p<0.01; significant difference between two groups (t₄ = 5.730, Student’s t-test). (F) Enhanced expression of Aldh3a1 in C/EBPα-downregulated 4T1 cells. C/EBPα was downregulated by miRNA. Values are the mean with SD (n = 3). The values of control group are set at 1.0. **p<0.01; significant difference between two groups (t4 = 4.892, Student’s t-test).