Tumor necrosis factor-α-induced protein-8 like-2 (TIPE2) upregulates p27 to
decrease gastic cancer cell proliferation
Supplemental Figures
Fig. 1
(A)
(B)
Fig. 1 Control immunohistochemistry of gastric cancers and adjacent tissues with no primary antibody. (A)
Gastric cancer tissues; (B) Adjacent tissues. Original magnification of Immunohistochemistry was ×200.
Fig. 2
(A)
(B)
Fig. 2 Comparison of the level of TIPE2 mRNA in paraneoplastic control and tumor tissues. (A) Gel electrophoresis of
the PCR products. The PCR templates used were as following: lane 1, no template; lane 2, cDNA of the mRNA
extracted from paraneoplastic control tissue; lane 3, cDNA of the mRNA extracted from gastric cancer tissue, the
cDNA was undetectable. (B) Quantitative real-time PCR analysis of TIPE2 mRNAs from adjacent paraneoplastic
control and tumor tissues (p=0.0047). The mRNA extration, cDNA synthesis, 35 cycles PCR and quantitative real-time
PCR were performed as described in the Materials and Methods.
Fig. 3
Homo sapiens tumor necrosis factor, alpha-induced protein 8-like 2 (TNFAIP8L2), mRNA
Sequence ID: ref|NM_024575.4|Length: 1199Number of Matches: 1
Range 1: 220 to 404GenBankGraphics Next Match Previous Match
Alignment statistics for match #1
Score
Expect Identities
Gaps
Strand
335 bits(181) 2e-89 184/185(99%) 1/185(0%) Plus/Plus
Query 10
GATGAGAC-AGCAGTGAGGTGCTAGATGAGCTCTACCGTGTGTCCAAGGAGTACACGCAC
|||||||| |||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 220 GATGAGACAAGCAGTGAGGTGCTAGATGAGCTCTACCGTGTGTCCAAGGAGTACACGCAC
279
Query
69
128
Sbjct
280
AGCCGGCCCCAGGCCCAGCGCGTGATCAAGGACCTGATCAAAGTGGCCATCAAGGTGGCT
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
AGCCGGCCCCAGGCCCAGCGCGTGATCAAGGACCTGATCAAAGTGGCCATCAAGGTGGCT
Query
129
GTGCTGCACCGCAATGGCTCCTTTGGCCCCAGTGAGCTGGCCCTGGCTACCCGCTTTCGC
188
68
339
Sbjct
340
Query
189
Sbjct
400
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
GTGCTGCACCGCAATGGCTCCTTTGGCCCCAGTGAGCTGGCCCTGGCTACCCGCTTTCGC
CAGAA
|||||
CAGAA
399
193
404
Fig. 3. The Basic Local Alignment Search Tool (BLAST) result of similarity between the sequences of PCR product
DNA bands.
Fig. 4
Fig. 4. TIPE2 expression was up-regulated with pRK5-tipe2 transfection in AGS cells. 1, AGS; 2, AGS transfected
with 2 μg pRK5-Mock; 3, AGS transfected with 2 μg pRK5-tipe2.
Fig. 5
(A)
Fig. 5. Original results of the Colony formation assays. (A) AGS; (B) BGC-823.
(B)
Fig. 6
(A) pRK5-mock
(B) pRK5-tipe2
Fig.6. Dot plots display cell apoptosis under the circumstance of TIPE2 restoration. (A) pRK5-mock plasmid
transfection; (B) pRK5-tipe2 plasmid transfection.
Fig. 7
(A) pRK5-mock
(B) pRK5-tipe2
Fig. 7. FACS histograms display cell cycle variation under the circumstance of TIPE2 restoration. (A) pRK5-mock
plasmid transfection; (B) pRK5-tipe2 plasmid transfection.
Fig. 8
Fig. 8. Quantification of activated Ras: whole-cell extracts from control and TIPE2 transfected AGS cells were assayed
for Ras activity using the Ras GTPase Chemi ELISA Kit. pRK5-tipe2 transfected AGS cells had significant difference
to the Mock plasmid transfected cells (p=0.0139), or to the cells with no transfection (p=0.0152).
Fig. 9
Fig. 9. Quantitative real-time PCR analysis of N-Ras and p27 mRNAs from adjacent paraneoplastic control and tumor
tissues). The mRNA extration, cDNA synthesis and quantitative real-time PCR were performed as described in the
Materials and Methods.
Fig. 10
Fig.10. p27 was involved in TIPE2-associated cell cycle arrest. 1, AGS with pRK5-tipe2 transfection; 2, AGS with
TIPE2 expression interfered by Control siRNA; 3, AGS with TIPE2 expression interfered by p27 siRNA; 4, AGS.
Western blot were performed as described in Materials and Methods.