Real-time PCR analysis
The total RNA of Huh7 cells transfected with empty vector or MIF4GD-myc
were extracted using a SuperfectRI reagents (Shanghai Pufei Biotechnologies). Then,
1 μg of total RNA was converted to cDNA using the First Strand cDNA Synthesis Kit
(Fermentas Scientific Inc). The primers used for real-time PCR are as follow: p27, 5’GAG CAG ACG CCC AAG AAG C-3’, and 5’- GTC CAT TCC ATG AAG TCA
GCG-3’; FanCd2, 5’-CCG GAA TAT TGG ATT CTC ACA T-3’, and 5’- GAA CTT
TCA CTC CTG GTC CAT C-3’; Coq5, 5’- TGC TCT ATG GAG CTA TTG CG-3’,
and 5’-TCA CAT CAT ACT TCT TAG CCA C-3’; Heatr1, 5’- CCA GGA ACG
GGT GAC AAA GC-3’, and 5’- CCT TTA GTT TTT CAG CCA GTG C-3’; Rbl2,
5’- ATT TGG CAT GGA AAC CAG AG-3’, and 5’-GTC CTC CAG TAT CAG
CAC GAA C-3’. β-actin, 5’-AGG CCA ACC GCG AGA AGA T-3’, and, 5’-TCA
CCG GAG TCC ATC ACG AT-3’.
5-Ethynyl-2′-deoxyuridine (EDU) cell proliferation assay
Bel-7404 cells were plated into 96-well plates with a density of 2×104 cell/well.
48 h after transfection, cells were incubated with 50 μM EdU in DMEM for 2 h. After
labeling, the cells were assayed with Cell-Light EdU DNA Cell Proliferation Kit
(Ribobio) in accordance with the manufacturer’s protocol. Cell proliferation was
analyzed using images from randomly photographed fields obtained on a CTR 5000
fluorescence microscope (Leica Microsystems, Bensheim, Germany). The assay was
performed using 6 wells for each group.
P27 translation assay
Huh7 cells were mock-transfected, or transfected with pCMV-Myc or
MIF4GD-Myc contracts. 24 h later, cells were incubated with methionine- and
cysteine-free DMEM (Invitrogen) for 30 min at 37°C and labeled with 20 µCi/ml
35S-methionine (PerkinElmer) in methionine- and cysteine-free DMEM containing
10% FBS for 20 min. The cells were harvested and lysed in a lysis buffer containing
50 mM Tirs-HCl, PH 7.5, 150 mM NaCl, 1% Triton X-100, 1% NP-40, 5 mM EDTA,
with Roche protease inhibitor cocktail and phosphatase inhibitor cocktail for 1 h at
4°C. Cell lysates were centrifuged at 13,000 rpm, 4°C for 15 min and the supernatant
were incubated with anti-p27 antibody at 4°C for overnight. Protein G-agarose was
added to precipitate the immunocomplexes at 4°C for 2 h. Finally, the
immunocomplexes were washed with lysis buffer for four times and subjected to
SDS-PAGE seperation followed by autoradiography.
Luciferase Based Histone RNA translation assay
The histone RNA translation assay experiment was carried out using a similar
strategy as previous reported (Neusiedler et al 2012). Briefly, Huh7 cells plated in
24-well plate were transfected with 20 ng poly(A)- or stem-loop-directed RNA
translation Luciferase constructs (pCL-6E1 and pCL-6E1.HSL) and 100 ng
pCMV-myc or MIF4GD-myc constructs (Wu et al 2006). 24 h after transfection, cells
were harvested and subjected to dual luciferase reporter assay using Dual Luciferase
Reporter Assay System (Promega) according to the manufacturer's protocol. Or
alternatively, the total RNA of the cells were extracted and reverse-transcribed into
cDNAs to examine the relative level of luciferase mRNA (β-actin as control) using
real time PCR analysis (primers, 5’-TCA AAG AGG CGA ACT GTG TG-3’, and
5’-GGT GTT GGA GCA AGA TGG AT-3’).
References:
Neusiedler J, Mocquet V, Limousin T, Ohlmann T, Morris C, Jalinot P (2012). INT6 interacts with
MIF4GD/SLIP1 and is necessary for efficient histone mRNA translation. RNA 18: 1163-1177.
Wu L, Fan J, Belasco JG (2006). MicroRNAs direct rapid deadenylation of mRNA. Proc Natl Acad Sci
U S A 103: 4034-4039.
SUPPLEMENTARY FIGURE AND TABLE LEGENDS
Supplementary Figure 1. MIF4GD has no significant influence on p27 mRNA
translation. Huh7 cells transfected with empty vector or MIF4GD-myc-expressing
vector were pulse labeled with 35S-methione. Then, p27 was immunoprecipitated
from the lysate and subjected to SDS-PAGE separation and autoradiography.
Supplementary Figure 2. MIF4GD enhances the transcription repression of p27
on its target genes. Bel-7404 cells were transfected with pCMV-myc or
MIF4GD-myc constructs. 48h after transfection, the total RNA of cells were extracted
and reverse-transcribed into cDNA. Real-time PCR were performed to determine the
mRNA level of indicated genes.
Supplementary Figure 3. MIF4GD didn’t alter p53 ubiquitination in HCC cells.
Huh7 cells were transfected with pCMV-myc or MIF4GD-myc. 24 h after transfection,
cells were treated with 20 μM MG132 for 6 h. The cells were lysed and
immunoprecipitated with anti-p53 antibody. The resulting immunoprecpitates were
blotted using anti-Ub antibody.
Supplementary Figure 4. MIF4GD decreases cell proliferation of HCC cells.
Bel-7404 cells mock-transfected, or transfected with empty or MIF4GD-myc
constructs were subjected to EDU incorporation assay. The relative percentage of
EDU-positive cells in each group were analyzed to indicate the grow potential of the
HCC cells.
Supplementary Figure 5. Knockdown of MIF4GD increases cells in S phase.
Huh7 (a) or HepG2 (b) cells were mock-transfected, or transfected with pSilencer 4.1,
shMIF4GD-1 or shMIF4GD-2 constructs. 48 h later, cells were subjected to flow
cytometrical analysis. Asterisks, P<0.05, significant difference between mock,
pSilencer 4.1 control groups and shMIF4GD groups.
Supplementary Figure 6. MIF4GD enhances the stem-loop luciferase reporter
activity. Huh7 cells transfected with pcDNA-myc or MIF4GD-myc as well as
pCL-6E1(polyA) or pCL-6E1.HSL(Stem-loop) luciferase reporter constructs were
analyzed for luciferase assay or the expression of luciferase mRNA level to determine
the effect of MIF4GD on translational activity of different types of 3’-end mRNA.
Supplementary Figure 7. MIF4GD over-expression in L02 hepatocytes didn’t
alter cellular p27 level. A. L02 hepatocytes mock-transfected, or transfected with
empty or MIF4GD-myc, were probed with p27 expression at 48 h after transfection. B.
48 h after transfection with MIF4GD-myc construct, L02 hepatocytes were
immunostained with mouse monoclonal anti-myc and rabbit polyclonal anti-p27
antibodies.
Supplementary Table 1. Relationship between MIF4GD, p27kip1 expression and
clinicopathological parameters in 108 HCC specimens.