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. 2019 Jul 23;20(9):e47987. doi: 10.15252/embr.201947987

Figure EV1. Characterization of c‐myc wt/wt and c‐myc ∆/∆ cells.

Figure EV1

  1. c‐myc copy number relative to a reference amplicon on the Ncl gene at different time‐points after LPS stimulation in c‐myc wt/wt and c‐myc ∆/∆ cells, as indicated. n = 6 for the t0 and 24 h samples, n = 4 for the 48‐h and 72‐h samples.
  2. c‐myc mRNA expression (normalized to TBP) at different time‐points after LPS stimulation in c‐myc wt/wt and c‐myc ∆/∆ cells. n = 3.
  3. Myc protein levels at different time‐points after LPS stimulation in c‐myc wt/wt and c‐myc ∆/∆ cells, based on quantification of four independent immunoblots (each produced with B‐cell lysates obtained from a pool of 3 mice per genotype): a representative blot is shown above the plot. Note that in wild‐type cells, c‐myc mRNA levels peaked 2 h after LPS stimulation 9, while the protein steadily accumulated over time, consistent with post‐transcriptional regulation of its synthesis and/or stability 63, 64: as expected, both mRNA and protein accumulation were blunted in c‐myc ∆/∆ cells.
  4. Myc and IgG ChIP, as indicated, in non‐treated (NT) or LPS‐treated (2 h) c‐myc wt/wt and c‐myc ∆/∆ B‐cells. PCR primers in the AchR promoter (as a non‐bound control) and in Ncl intron 1 (as a known Myc target with 5 E‐boxes) were used for quantification (% if input) as previously described 39, 65. n = 3.
  5. Quantification of total RNA levels per cell along the LPS time‐course in c‐myc wt/wt and c‐myc ∆/∆ cells. n = 3.
  6. Percentages of BrdU‐positive cells at 0, 12, and 24 h, as indicated. n = 3.
  7. Growth curve of c‐myc wt/wt and c‐myc ∆/∆ cells, as indicated. n = 3.
  8. c‐myc wt/wt and c‐myc ∆/∆ cells were sorted according to their size and shape (FSC and SSC, respectively) after 24 or 48 h of LPS stimulation: cells with low and high FSC + SSC were defined as resting and activated, respectively, and were purified by FACS sorting prior to qPCR quantification of c‐myc copy number, alongside unsorted c‐myc ∆/∆ and c‐myc wt/wt control samples. Results from a representative experiment are shown. The experiment was repeated twice with similar results.
  9. Caspase‐3/7 activity normalized on cell numbers along the LPS time‐course in c‐myc wt/wt and c‐myc ∆/∆ cells. n = 3.
Data information: In all the bar plots (except panel H), data are presented as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, Student's t‐test.