SUPPLEMENTARY INFORMATION
Supplementary Fig. 1. Control of hRFPL1 expression and activity. (A) Impact of p53
RNA interference on Pax6-regulated genes, FOXM1 and MCM5, was assessed by quantitative
RT-PCR. *P<0.05 vs. GFP/control shRNA cells using one-way ANOVA followed by Tukey
post-hoc test. (B) Impact of p53 gain of function in different cell lines on hRFPL1 and
CDKN1A expression determined by quantitative RT-PCR. (C) Impact of Pax6 gain of
function in different cell lines on hRFPL1 determined by quantitative RT-PCR. In (B) and
(C), CT values for hRFPL1 were not different from those of RT-negative samples except in
Pax6-expressing HeLa cells. (D) Impact of hRFPL1 gain of function on cell number of
different cell lines three days after transduction. *P<0.05, **P<0.01, ***P<0.001 vs. eGFP
cells using Student’s t-test. (C) Impact of p53 RNA interference on Pax6-regulated genes,
FOXM1 and MCM5, was assessed by quantitative RT-PCR. *P<0.05 vs. GFP/control shRNA
cells using one-way ANOVA followed by Tukey post-hoc test. (E) Cellular expression of
FLAG-tagged hRFPL1 and its different FLAG-tagged truncated forms was performed by
immunofluorescence using a FLAG antibody two days after transduction. (F) Quantification
of the level of phospho-hRFPL1-FLAG proteins from immunoblotting experiments using a
FLAG antibody in DMSO- (control), Cdc2 inhibitor- or Gö6983- (PKC inhibitor) treated
HeLa cells. Normalization was performed using the amount of the actin reference protein in
each condition and band intensities were measured using the Image J software. *P<0.05 vs.
eGFP cells using one-way ANOVA followed by Tukey post-hoc test.
Supplementary Fig. 2. Impact of hRFPL1 on cyclin B1 and Cdc2 protein levels. (A)
Quantification of the levels of cell-cycle associated proteins in eGFP- or hRFPL1-expressing
HeLa cells from immunoblotting experiments using the indicated antibodies. Normalization
was obtained using the amount of the actin reference protein in each condition and band
1
intensities were measured using the Image J software. **P<0.01, ***P<0.001 using Student's
t-test. (B) Quantification of cyclin B1 and Cdc2 protein levels from immunoblotting
experiments in eGFP- or hRFPL1-expressing HeLa cells treated either with DMSO (control)
or MG132, a proteasome inhibitor. Normalization was performed using the amount of the
actin reference protein in each condition and band intensities were measured using the Image
J software. *P<0.05 vs. control cells using one-way ANOVA followed by Tukey post-hoc
test. (C) Quantification of cyclin B1 and Cdc2 protein levels from immunoblotting
experiments in HeLa cells expressing eGFP, WT hRFPL1, S200A, or S287E hRFPL1
mutants. Normalization was performed using the amount of the actin reference protein in each
condition and band intensities were measured using the Image J software. *P<0.05 vs. eGFP
cells using one-way ANOVA followed by Tukey post-hoc test. (D) Time course of the
proliferation of HeLa cells transduced with GFP, hRFPL1, hRFPL2, hRFPL3 or mRFPL.
***P<0.001 vs. GFP cells using two-way ANOVA followed by Tukey post-hoc test. (E)
Levels of cyclin B1 and Cdc2 proteins in GFP-, hRFPL1-, hRFPL2, hRFPL3- or mRFPLtransduced HeLa cells were determined by immunoblotting. (F) Quantification of cyclin B1
and Cdc2 protein accumulation over time from immunoblotting experiments in eGFP- or
hRFPL1-expressing HeLa cells following release (time 0) from mimosine-induced G1-phase
block. Normalization was performed using the amount of the actin reference protein in each
condition and band intensities were measured using the Image J software. **P<0.01,
***P<0.001 using two-way ANOVA followed by Tukey post-hoc test.
2
Supplementary Table I. Oligonucleotide probes and conditions used for quantitative
RT-PCR and semi-quantitative end-point RT-PCR
Gene
hRFPL1 (qPCR)
hRFPL1
(promoter)
hRFPL1 (exon)
CCNB1 (qPCR)
CDC2 (qPCR)
TP53
CDKN1A
CDKN1A
(promoter)
CDKN1A (exon)
L32
Primer sequences
F: CTg AAg gTg gTT CCC ATg TCT AT
R: gAC TTg gAg gAg CAA AAA ACA AgT
F: ACA CTg TCA ggA TgA Agg ATT T
R: AAA CAC ACA CAC Agg ATg g
F: gAA CAA AAT CAg gCC CAg TT
R: CTT ggA ggA gCA AAA AAC AAg Tg
F: CgC AAA gCg CgT TCC T
R: AAT gAC TTT TCC AgT AgC TgA Agg TTT
F: CTA gCA TCC CAT gTC AAA AAC TTg
R: CAg TgC CAT TTT gCC AgA AA
F: CCT CAC CAT CAT CAC ACT gg
R: TCT gAg TCA ggC CCT TCT gT
F: gAg CgA Tgg AAC TTC gAC TT
R: ggC gTT Tgg AgT ggT AgA AA
F: ATg TAT Agg AgC gAA ggT gCA gAC
R: CCT CCT TTC TgT gCC TgA AAC A
F: gAg CgA Tgg AAC TTC gAC TT
R: ggC gTT Tgg AgT ggT AgA AA
F: gTg AAg CCC AAg ATC gTC AA
R: TTg gTg ACT CTg ATg gCC Ag
3
Amplicon
size
Tm
#
cycles
81
-
-
264
57
40
600
57
40
157
-
-
102
-
-
426
58
28
328
58
28
377
57
40
1531
57
40
249
58
26