Fig 2: Expression analysis of mnt+/+ and mnt+/− lymphoid cells from WT and myc transgenic mice. (a) Mnt and transgenic MYC protein expression in thymocytes from mnt+/+ and mnt+/− WT and vavP-MYC17 mice. Western blots of lysates of thymocytes from four-week-old mice of the indicated genotypes, probed with antibodies specific for Myc, Mnt, Max and β-actin (see Materials and Methods). Each lane represents an individual mouse. Comparable results were obtained for thymocytes from mnt+/+ and mnt+/− vavP-MYC10 mice. (b) Mnt and transgenic Myc protein expression in pre-B cells from mnt+/+ and mnt+/− Eμ-myc mice. Western blot analysis of pre-B cells (B220+sIg−) sorted by flow cytometry from bone marrow of 4-week-old mice of indicated genotypes. Each lane represents an individual mouse and MWs (kD) of markers run in parallel are indicated. Endogenous Myc was not detectable in WT control cells. (c) Quantification of Mnt and Myc protein expression in mnt+/+ and mnt+/− WT and Eμ-myc mice. The blot shown in (b) and two additional blots using independent samples were quantified using ChemiDoc and Image Lab software (Bio-Rad), with normalisation to actin expression. Expression is relative to the first mnt+/+ Eμ-myc sample on each blot. (d) Quantitative PCR analysis of myc and mnt RNA in pre-B cells isolated using magnetic beads coated with CD19 antibody from the bone marrow of 4-week-old mice of the indicated genotypes (n=2–15). Data are normalised to myc expression in mnt+/+ Eμ-myc mice

Fig 3: MAX repression of clock genes requires MNT. (A) Expression of clock genes PER1, PER2, CRY1 and CRY2 in MDA-MB-231 cells upon the knockdown of MAX (siMAX), MNT (siMNT). Relative expression was determined by qRT–PCR using GAPDH for normalization. Values of control cells were set to 1. Shown as mean + SEM, n ≥ 3. *** p < 0.001 and ### p < 0.001, two-way ANOVA with Bonferroni post hoc test, siMAX or siMNT versus control cells, respectively. (B) Protein levels of PER2, MNT and MAX in MDA-MB-231 cells transfected with siRNA sequences against MAX (siMAX), MNT (siMNT) or a non-targeting control. GAPDH was used as a loading control. (C) Recruitment of MNT on the promoters of PER2, CRY1 and CRY2 in MAX-silenced (siMAX) and control MDA-MB-231 cells. Enrichment of MNT was evaluated by quantitative PCR of immunoprecipitated DNA compared with input DNA (% of input). Immunoglobulin G (IgG) was used as a negative control. Shown as mean + SEM, n =3. ** p < 0.01 and *** p < 0.001, two-way ANOVA with Bonferroni post hoc test, α-MNT precipitated DNA from siMAX versus control chromatin samples. (D) Chromatin samples from C (MAX-silenced and control) were immunoprecipitated with α-MYC. Enrichment of MYC on the MYC-target NCL promoter was evaluated by quantitative PCR of immunoprecipitated DNA compared with input DNA (% of input). Immunoglobulin G (IgG) was used as a negative control. Shown as mean + SEM, n =3. (E) Expression of clock genes PER1, PER2, CRY1 in MCF10A cells knocked down for MNT (siMNT). Relative expression was determined by qRT–PCR using GAPDH for normalization. Values of control cells were set to 1. Shown as mean + SEM, n ≥ 3. * p < 0.05, ** p < 0.01 and *** p < 0.01, two-way ANOVA with Bonferroni post hoc test, silenced versus control cells. (F) Real-time bioluminescence oscillatory pattern in MNT-silenced (siMNT, green dots) and control (black dots) MCF10A cells expressing a firefly luciferase reporter controlled by the circadian-responsive BMAL1 promoter. Shown as baseline-subtracted luminescence data fitted to a sine wave.

Fig 4: Analysis of the effect of modulating miR-378a-3p levels upon the protein levels of the target genes. Representative examples of the effect of miR-378a overexpression on IRAK4 (A), FOXP1 (C), and MNT (E). Representative examples of the effect of miR-378a-3p inhibition on IRAK4 (B), FOXP1 (D), and MNT (F). cells. Graphs show the quantification of the protein levels of three to four independent infections. For each protein, the part of the total protein lane corresponding to the position of the protein band is shown as an indication for protein loading. Protein levels were quantified relative to the total protein amount as measured in the complete lane. Uncropped blots can be found in Figure S6. Significant differences were calculated using a paired t-test. * p < 0.05, ** p < 0.01, ns = not significant.

Fig 5: Overexpression of IRAK4 and MNT phenocopies the effect of miR-378a-3p inhibition in DG75 cells. (A) Validation of the IRAK4 and MNT overexpression in DG75. An empty vector (EV) was used as a control. For each protein, the part corresponding to the position of the protein band is shown as control of protein loading. Protein levels were quantified relative to the total protein amount in the complete lanes. Uncropped blots can be found in Figure S7. (B) GFP competition assays upon overexpression of IRAK4 and MNT resulted in a strong decrease in GFP+ cells over time, while no or only a mild effect was observed for the EV control. The GFP percentages were measured for 11 days, and the GFP percentage as measured on day 4 was set to 100%. Assays were performed in duplicate. Significant differences between IRAK4 and MNT overexpression and EV control were calculated using a mixed model analysis. ** p < 0.01 and **** p < 0.0001.