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HEP_25834_sm_SuppFig1.tif3738KSupporting Information Figure. S1. Levels of KIAA0101 tv1 mRNA were up-regulated in human HCC tissues. (A) Semi-quantitative RT-PCR showed the high level of KIAA0101 tv1 mRNA in HCCs and its expression was weak in NT. Four pairs of HCC samples were exhibited here. (B) The graph shows the mean±standard error of KIAA0101 tv1 mRNA levels by semi-quantitative RT-PCR. **P<0.005. (C) Increased expression of KIAA0101 tv1 in HCC (lane 1) was detected by virtual northern blot analysis. And its expression was hardly detectable in NT (lane 2). GAPDH was used as control. Lane 3 is a marker. (D) The graph shows the mean±standard error of KIAA0101 tv1 mRNA levels by virtual northern blot. **P<0.005.
HEP_25834_sm_SuppFig2.tif9946KSupporting Information Figure. S2. Exogenous overexpression of KIAA0101 tv1 induced transformation of NIH3T3. NIH3T3 cells transfected with KIAA0101 tv1 (NIH3T3-K) or vector (NIH3T3-(-)) were selected with G418. (A) RT-PCR and western blot analysis. The resistant clones were analyzed by KIAA0101 tv1-specific primers or antibody. HepG2 was used as positive control. β-actin was used as a loading control. (B and C) Colony formation assay. NIH3T3-K cells resulted in significant increase of the numbers of colonies, comparing with the NIH3T3-(-) cells (B). The graph shows the mean±standard deviation of the colony number based on three independent experiments. **P<0.005 (C). (D and E) Tumor formation in nude mice. NIH3T3, NIH3T3-(-) and NIH3T3-K cells were inoculated into the flank of 6-week-old nude mice. After 4 weeks, NIH3T3-K clones formed a large mass at the right flank of nude mice. HepG2 was used as positive control (D). The growth curve was measured by the tumor volume of each five days after injection. It shows the mean±standard deviation of tumor volume based on five independent experiments. **P<0.005 (E).
HEP_25834_sm_SuppFig3.tif760KSupporting Information Figure. S3. KIAA0101 tv1 prevented HepG2 and NIH3T3 cells from ADR-induced apoptosis. Cell survival assays. HepG2 cells (A and B) or NIH3T3 cells (C and D) were transfected with either the plasmid encoding the KIAA0101 tv1 (NIH3T3-K) or an empty vector (NIH3T3-(-)), and then treated with serial dilutions of ADR. 48 hours post ADR treatment, cell viability was determined by MTT assays. The cell viability is shown as the mean±standard deviation based on five independent experiments. *P<0.05, **P<0.005, ***P<0.001.
HEP_25834_sm_SuppFig4.tif15209KSupporting Information Figure. S4. shRNA mediated down-regulation of the endogenous KIAA0101 tv1 promoted ADR-induced apoptosis in HepG2 cells. HepG2 cells were transiently transfected with shRNA against KIAA0101 tv1 (HepG2-pShK), vector control (HepG2-pSh(-)) or scrambled shRNA (HepG2-pShCntrl). 48 hours after transfection, cells were treated as indicated. (A) Check the level of KIAA0101 tv1. mRNA were prepared and processed for semi-quantitative RT-PCR with primers for KIAA0101 tv1 (upper panel). β-actin was also analyzed as a control (lower panel). (B) Cell survival assays. The cell survivals were examined by MTT assay after treatment with different concentration of ADR for 48 hours. The cell viabilities are shown as the mean±standard deviation based on four independent experiments. (C, D and E) Cell apoptosis assay by flow cytometry. Cell apoptosis was detected with Annexin V-FITC/propidium iodide after ADR treatment (C). The apoptotic cells are shown as the mean±standard deviation based on three independent experiments. *P<0.05 (D and E). (F) Knockdown of p53. HepG2 cells were transiently transfected with shRNA against p53 (HepG2-pShp53), vector control (HepG2-pSh(-)) or scrambled shRNA (HepG2-pShCntrl). Knockdown effect was tested with semi-quantitative RT-PCR.
HEP_25834_sm_SuppFig5.tif2151KSupporting Information Figure. S5. Exogenous expression of KIAA0101 tv1 inhibited the activity of p53 in response to ADR treatment. (A) HepG2 cells were transiently transfected with 2 μg of the empty plasmid (HepG2-(-)) or with the expression plasmid for KIAA0101 tv1 (HepG2-K). 48 hours after transfection, cells were treated with 0.6 μM ADR. After another 48 hours treatment, total mRNA were also prepared and subjected to quantitative RT-PCR using specific primers. β-actin was used as control. The experiments were performed at least three times. (B) The graph shows the mean±standard deviation of mRNA levels by real-time RT-PCR based on three independent experiments. *P<0.05, **P<0.05.
HEP_25834_sm_SuppInfo.rtf79KSupporting Information
HEP_25834_sm_SuppTables1_4.doc38KSupporting Information Tables.

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