Relationship between the expression of Bmi1 and levels of TAMs.

<p>(<b>A</b>) Immunohistochemistry of Bmi1, CD68, and CD163 expression in 83 gastric cancer tissues. Scale bars, 100um. (<b>B</b>) The percentage of CD68/163 positive specimens in high Bmi1 expressing gastric cancer. There was a significant correlation between Bmi1 expression and CD68/163 expression (*<i>P</i> < 0.05, ***<i>P</i> < 0.001, respectively). (<b>C</b>) Immunohistochemistry of Bmi1, CD68, and CD163 expression in 49 colon cancer tissues. Scale bars, 100um. (<b>D</b>) The percentage of CD68/163 positive specimens in high Bmi1 expressing colon cancer. There was a significant correlation between these two groups (**<i>P</i> < 0.01, **<i>P</i> < 0.01, respectively).</p

Bmi1 expression and sphere assay in gastrointestinal cancer cell lines co-cultured with M1- or M2-polarized THP-1 macrophages.

<p>(<b>A</b>) Cytokine production profile of M1- and-M2 polarized THP-1 macrophages. (<b>B</b>) qRT-PCR analysis of Bmi1 expression in AGS cells co-cultured with M1- and M2-polarized THP-1 macrophages, compared with Bmi1 expression in AGS cells only as a control group. Significantly higher Bmi1 expression was detected in co-cultured groups compared with the control group (***<i>P</i> < 0.001, ***<i>P</i> < 0.001, respectively). (<b>C</b>) qRT-PCR analysis of Bmi1 expression in HCT116 cells co-cultured with M1- and M2-polarized THP-1 macrophages, compared with Bmi1 expression in HCT116 cells only as a control group. Significantly higher Bmi1 expression was detected in co-cultured groups compared with the control group (***<i>P</i> < 0.001, ***<i>P</i> < 0.001, respectively). (<b>D</b>) Microscopic images of 3D sphere cultured AGS cells co-cultured with macrophages, compared with 3D sphere cultured AGS cells only as a control group. Scale bars, 100um. (<b>E</b>) Microscopic images of 3D sphere cultured HCT116 cells co-cultured with macrophages, compared with 3D sphere cultured HCT116 cells only as a control group. Scale bars, 100um. (<b>F</b>) Significantly higher sphere numbers were detected in co-cultured groups compared with the control group in AGS cells (*<i>P</i> < 0.05, *<i>P</i> < 0.05, respectively). (<b>G</b>) Significantly higher sphere numbers were detected in co-cultured groups compared with the control group in HCT116 cells (*<i>P</i> < 0.05, **<i>P</i> < 0.01, respectively).</p

miR-30e* suppresses Bmi1 expression in gastrointestinal cells.

<p>(<b>A</b>) Western blot analysis of Bmi1 expression in 6 gastrointestinal cancer cell lines. (<b>B</b>) Western blot analysis of Bmi1 expression in high Bmi1-expressing AGS cell lines transfected with negative control (NC) and miR-30e* mimics. (<b>C</b>) Western blot analysis of Bmi1 expression in high Bmi1-expressing HCT116 cell lines transfected with NC and miR-30e* mimics. (<b>D</b>) Western blot analysis of Bmi1 expression in low Bmi1-expressing NUGC4 cell lines transfected with NC and miR-30e* inhibitors. (<b>E</b>) Western blot analysis of Bmi1 expression in low Bmi1-expressing COLO201 cell lines transfected with NC and miR-30e* inhibitors.</p

miR-30e* expression in human gastrointestinal cancer tissues.

<p>(<b>A</b>) The levels of miR-30e* expression in 16 gastric cancer tissues and their matched adjacent normal gastric epithelia as assessed by qRT-PCR. (<b>B</b>) The levels of miR-30e* expression in 29 of high and 24 of low Bmi1-expressing gastric cancer tissues as assessed by qRT-PCR. (<b>C</b>) The levels of miR-30e* expression in 37 colon cancer tissues and their matched adjacent normal colon epithelia as assessed by qRT-PCR. (<b>D</b>) The levels of miR-30e* expression in 20 of high and 17 of low Bmi1-expressing colon cancer tissues as assessed by qRT-PCR.</p

miR-30e* downregulates Bmi1 expression by directly targeting its 3′ UTR.

<p>(<b>A</b>) 3D sphere culture grown in serum-free non-adherent culture with AGS cells co-cultured with macrophages and transfected with mimic miR-30e*, compared with 3D sphere culture with AGS cells co-cultured with macrophages and transfected with mimic NC as a control group. Scale bars, 100um. (<b>B</b>) Significantly low sphere numbers were detected in mimic miR-30e* transfected groups compared with the control group (*P < 0.05, *P < 0.05, respectively). (<b>C</b>)The putative miR-30e* target site or a mutated sequence of the 3′ UTR of Bmi1 was cloned immediately downstream of the luciferase gene. (<b>D</b>) Luciferase activity of AGS cells co-transfected with plasmids containing the wild-type miR-30e* target sequence in the 3′ UTR of Bmi1 or control plasmids along with the mRNA mimic NC and mimic miR-30e*. (<b>E</b>) Luciferase activity of AGS cells co-transfected with plasmids containing the wild-type or mutant miR-30e* target sequence in the 3′ UTR of Bmi1 along with the mRNA mimic NC and mimic miR-30e*. </p

Expression of miR-30e* and Bmi1 co-cultured with M1- and M2-polarized macrophages purified from human monocytes.

<p>(<b>A</b>) qRT-PCR analysis of miR-30e* expression in AGS cells co-cultured with M1- and M2-polarized macrophages. Significantly lower miR-30e* expression was detected in co-cultured groups compared with the control group (***<i>P</i> < 0.001, *<i>P</i> < 0.05, respectively). (<b>B</b>) qRT-PCR analysis of miR-30e* expression in HCT116 cells co-cultured with M1- and M2-polarized macrophages. Significantly lower miR-30e* expression was detected in co-cultured groups compared with the control group (***<i>P</i> < 0.001, **<i>P</i> < 0.01, respectively). (<b>C</b>) qRT-PCR analysis of Bmi1 expression in AGS cells co-cultured with M1- and M2-polarized macrophages. Significantly higher Bmi1 expression was detected in co-cultured groups compared with the control group (***<i>P</i> < 0.001, **<i>P</i> < 0.01, respectively). (<b>D</b>) qRT-PCR analysis of miR-30e* expression in HCT116 cells co-cultured with M1- and M2-polarized macrophages. Significantly higher Bmi1 expression was detected in M1 macrophage co-cultured groups compared with the control group (**<i>P</i> < 0.01). (<b>E</b>) Schematic representation of miR-30e*-Bmi1 signaling mediated by TAMs. </p