Shao-Ming Shen et al Role of I in MT of cancers MO reports xpanded View igures igure V1. nalysis of the expression of I isoforms in cancer cells and their interaction with PTN. RT PR detection of Ish and Ish2 mrn with primers I9 and R4 (left), and Ish3 mrn with primers SH31 and HR1 (right) in SW620 cells. Western blots with an antibody specifically against residues 151 168 of I in the indicated cells. mpty arrowheads point to unknown bands. lag-tagged I isoforms were cotransfected with H-tagged PTN into 293T cells, followed by co-ip with M2 beads. Western blots were performed for the indicated proteins. rrows point to the indicated lag-tagged I isoforms. igure V2. I regulates PTN oxidation. Western blots for recombinant PTN prepared under non-reducing or reducing condition. ox indicates oxidized protein. The recombinant PTN protein (50 ng) was preincubated with or without 2 mm H 2 O 2 for 10 min, followed by incubation with recombinant I (50 ng) in the presence of 200 lm NH at 4 for 1 h. The mixtures were prepared under non-reducing or reducing condition and blotted for the indicated proteins. ox indicates oxidized protein and re indicates the reduced band of PTN. The recombinant PTN protein (50 ng) was incubated with recombinant I or its deleted mutant INH (50 ng) in the presence of 200 lm NHat4 for 1 h. The mixtures were prepared under non-reducing or reducing condition and blotted for the indicated proteins. ox indicates oxidized protein and re indicates the reduced band of PTN. Western blots for the indicated proteins of shn- or shi-infected SW620 cells., ell lysates from shn- or shi-infected HT29 () or U145 () cells were prepared under reducing or non-reducing condition and analyzed by Western blots for the indicated proteins. Phosphorylated kt and GSK-3b in U145 cells were quantified and normalized to total kt and GSK-3b, respectively. ox indicates oxidized protein and re indicates reduced PTN. G ell lysates from P3 (left) and LNaP (right) cells with or without I knockdown were analyzed by Western blots for the indicated proteins. Phosphorylated kt and GSK-3b were quantified and normalized to total kt and GSK-3b, respectively. H, I shn- and shi#1-infected SW620 cells were treated with 50 lm H 2 O 2 for the indicated times (H) or with a gradient of H 2 O 2 (I) and subjected to Western blots for the indicated proteins under non-reducing condition. ox indicates oxidized protein and re indicates reduced PTN. J luorescence intensity of staining of shn- or shi-infected SW620 cells was analyzed by flow cytometry (left), and quantified data were shown (right). ata represent means and s.d of three independent experiments. Two-sided unpaired t-test. K SW620 cells were treated with or without 100 lm H 2 O 2 for 30 min and subjected ro immunofluorescence staining of I with re-staining of PI. Scale bar representing 20 lm. L lag-ptn-expressing SW620 cells treated with or without 100 lm H 2 O 2 were subjected to co-ip with anti-lag antibody and analyzed by Western blots for the indicated proteins. M SW620 cells were treated with 100 lm H 2 O 2 for the indicated times in the absence or presence of N (10 mm) pretreatment. Lysates were prepared under reducing or non-reducing condition and analyzed by Western blots for the indicated proteins. ox indicates oxidized protein and re indicates reduced PTN. N V-, lag-tagged PTN-WT-, and PTN-71S-expressing cells were subjected to co-ip with anti-lag antibody and analyzed by Western blots for the indicated proteins. O V-, lag-tagged PTN-WT-, and PTN-71S-transfected P3 cells were treated with 100 lm H 2 O 2 for 30 min. Lysates were prepared under reducing or nonreducing condition and analyzed by Western blots for the indicated proteins. ox indicates oxidized protein and re indicates reduced PTN. * indicates a nonspecific band. ª 2015 The uthors MO reports V1
MO reports Role of I in MT of cancers Shao-Ming Shen et al G H J I K L M N O igure V2. V2 MO reports ª 2015 The uthors
Shao-Ming Shen et al Role of I in MT of cancers MO reports igure V3. I regulates WNT/b-catenin signaling target genes. The folds of relative mrn levels of the indicated genes against shn-infected cells were quantified by quantitative real-time PR in shi#1-infected HT29 cells. Western blots for xin2 and b-actin of shn- or shi-infected SW620 cells. V, I, or Ish3 was transfected into shn- or shi-infected SW620 cells. The folds of relative mrn levels of the indicated genes against V-transfected shn cells were quantified by quantitative real-time PR. Quantitative real-time PR for mrn levels of the indicated genes in shn- or shi-infected SW620 cells with or without b-catenin knockdown. ata information: In (,, ), data represent means and s.d of three independent experiments. Twosided unpaired t-test. ª 2015 The uthors MO reports V3
MO reports Role of I in MT of cancers Shao-Ming Shen et al igure V4. I silence induces MT in cancer cells and correlation between I and -cadherin in colon cancer tissues., The morphology (left, scale bar, 100 lm) and Western blots for the indicated proteins (right) of HT29 cells () and U145 cells (). Representative IH images of two cases of colon cancer samples stained with I or -cadherin. Right panels show the enlarged picture of the boxed area. Pearson correlation between the IRS scores of I and -cadherin. Two-sided unpaired t-test. ox plots of -cadherin expression in colon cancer tissues from 74 subjects with low and high I expression. Horizontal lines represent the median; the bottom and top of the boxes represent the 25 th and 75 th percentiles, respectively; and the vertical bars represent the range of data. ny outliers are marked with a circle. ata was analyzed using Mann Whitney U-test. Western blots for the indicated proteins of shn- or shi-infected SW620 cells. V4 MO reports ª 2015 The uthors
Shao-Ming Shen et al Role of I in MT of cancers MO reports G H I J K L igure V5. ata in xenografts and cancer patients. Representative image of tumors developed in a mouse that received subcutaneous injection of shn- and shi#1-infected SW620 cells. Tumor growth curves after subcutaneous injection of shn- or shi#1-infected SW620 cells. ata represent means and s.d of five mice in each group. Western blots for the indicated proteins in subcutaneous growth derived from shn- or shi#1-infected SW620 cells. Representative images of colon tumors derived from the subcutaneous engraftments of shn- or shi#1-infected SW620 cells. olon tumors derived from the subcutaneous engraftments were sectioned and stained with hematoxylin and eosin (H&). Scale bar represents 100 lm., G Ten general metastasis-related genes as described in the text were evaluated in I knockdown () or overexpressing (G) SW620 cells. ata represent means and s.d of three independent experiments. Two-sided unpaired t-test. H Representative images show the formation of tumors by splenic injection of shn-, shi#1-, and shi#2-infected HT29 cells in the mouse. I Representative IH images of two cases of colon cancer samples. Scale bar, 100 lm. J The numbers of cases with I low and I high expression in our cohort. K, L Kaplan Meier estimates of overall survival of subjects with high and low I expressions in TG reast Invasive arcinoma (K) and TG Lung Squamous ell arcinoma (L). ª 2015 The uthors MO reports V5