Fig 2: chARF and p16-ACT arise from a CC to T mutation in exon 2 of INK4A.(A) p16 and p14ARF are initiated from exons 1a and 1ß, respectively, but share exons 2 and 3. The CC to T mutation found in the M2 cell line is shown by a black bar. Patterned areas represent respective coding regions. (B) Comparison of wild-type p14ARF and p16 transcripts versus the chARF and p16-ACT transcripts derived from M2 cells. Reading frames are noted by single bold lines. Codons 94 and 81 are boxed in bold for p14ARF and p16, respectively. The reading frame of chARF is switched from the p14ARF reading frame to the p16 reading frame after codon 94 of p14ARF, codon 81 of p16. At the same respective codon, the reading frame of p16-ACT changes from a p16 reading frame to a non-p16/non-p14ARF reading frame. (C) The proteins resulting from wild-type and M2 INK4A transcripts are shown for comparison. chARF contains the amino terminus (N-terminus) of p14ARF and the carboxyl terminus (C-terminus) of p16. p16-ACT contains the amino terminus of p16 with a non-p16/non-p14ARF carboxyl terminus. (D) Western blot of chARF and p16-ACT in M2 cells. U2OS cells were transfected with chARF or p16-ACT and used as controls. ß-actin is shown as a control for loading differences. chARF protein was detectable in M2 cells while p16-ACT was undetectable.

Fig 3: Staufen1 has differential roles in ERMS and ARMS via c-myc-dependent and -independent events, respectively.Western blot analysis of ERMS (RD) and ARMS (RH30) cells following 48 hours of Control (CTL) or Staufen1-shRNA (shStau1) expression for (a) Staufen1 expression (b) c-myc expression and (c) p14ARF. All quantifications are normalized to ß-actin and represented as a percentage of the Control (CTL) with n = 3. Polysome profiling of (d) ERMS (RD) and (e) ARMS cells expressing CTL or shStau1. Polysome profile obtained by continuous absorbance readings at 254 nm (Top) and c-myc mRNA expression measured by qRT-PCR from 10 × 1 mL sucrose gradient fractions (Bottom). Data are Mean ± SEM, *P < 0.05, **P < 0.01, ***P < 0.001.

Fig 4: chARF slows the growth of U2OS cells.(A) U2OS cells were transfected with p16, p16-ACT, p14ARF, chARF, or GFP control. After 48 hours, cells were counted using Trypan blue to assess cell number and viability. p14ARF and chARF reduced cell growth by 73% compared to GFP control cells (p = 0.002 two-tailed unpaired T-test compared to GFP control) while p16 reduced growth by 46% (p = 0.02 two-tailed unpaired T-test compared to GFP control). p16-ACT had no significant effect on growth (p = 0.8 two-tailed unpaired T-test compared to GFP control). (B) Cells were transfected with p16, p16-ACT, p14ARF, chARF, empty vector (Vector), or a negative control vector without the G418 selection gene (Negative Control) and grown in 400 µg/ml G418 for four weeks. Colonies were the stained and counted. Percent colony formation is expressed as a percent of the vector control. p14ARF and chARF reduced colony formation by 86% and 84%, respectively, compared to vector control (p = 0.006 two-tailed unpaired T-test). p16 was slightly less potent, slowing colony formation by 75% (p = 0.008 two-tailed unpaired T-test compared to vector control) while p16-ACT slowed colony formation by 31% (p = 0.04 two-tailed unpaired T-test compared to vector control). Right: the average of three replicate experiments with standard deviation. *p<0.05 two-tailed unpaired T-test. Left: plates of a representative experiment showing stained colonies.

Fig 5: chARF arrests U2OS cells in the G1 and G2/M phases of the cell cycle.U2OS cells were transfected with p16, p16-ACT, p14ARF, chARF, GFP, or no DNA (mock). 24 hours after transfection, cells were (A) left untreated or (B) treated with 100 ng/ml nocodazole to induce G2/M arrest, highlighting any G1 maintenance while distinguishing between G1 arrest versus shortening of the S and G2/M phases. 48 hours after transfection, cells were fixed, stained for total DNA content using propidium iodide and analyzed by flow cytometry. Cell cycle distribution was determined using FlowJo analysis software. Histograms show the G1 and G2/M peaks (2 n diploid and 4 n quadraploid cells, respectively) along with percent of relative distribution frequencies. p16 transfected cells showed a stark G1 arrest when untreated and were resistant to G2/M arrest by nocodazole. p14ARF and chARF transfected cells had identical cell cycle distributions, showing G2/M arrest when untreated and maintenance of G1 levels when nocodazole treated. The cell cycle distribution of p16-ACT transfected cells was indistinguishable from that of the controls under both untreated and nocodazole treated conditions. Values shown are the average of two replicate experiments with standard deviation. *p<0.05 Wald's test. Histograms shown are from a representative experiment.