SMCP knockdown abrogates tumor initiation of LHK2 cells. A. Downregulation of SMCP expression in LHK2 cells transfected with siRNA. B. Representative picture of tumors injected with SMCP knockdown LHK2 cells. C. Tumor growth of LHK2, A549 and Lc817 cells transfected with SMCP siRNAs.

<p>Two×10⁵ LHK2 cells transfected with control siRNA or SMCP siRNA 1 and 2 were injected into NOD/SCID mice subcutaneously. One×10⁴ A549 cells transfected with control siRNA or SMCP siRNA2 were injected into NOD/SCID mice subcutaneously. One×10⁴ Lc817 cells transfected with control siRNA or SMCP siRNA2 were injected into NOD/SCID mice subcutaneously. Data are means + SD. Asterisks represents statistical significant difference. Student's t-test. <i>P</i><0.05.</p

SMCP knockdown abrogates tumor initiation of LHK2 SP cells.

<p>LHK2 SP cells were isolated and seeded onto a 24-well plate. SMCP siRNA was transfected into LHK2 SP cells. Two days after transfection, LHK2 SP cells were injected into NOD/SCID mice. Data are means + SD. Asterisks represents statistical significant difference. Student's t-test. <i>P</i><0.05.</p

Isolation of a novel CSC/CIC molecule, SMCP.

<p><b>A. Isolation of SP cells from LHK2, MCF7 and SW480 cells.</b> Lung (LHK2), colon (SW480) and breast (MCF7) cancer cells were stained with Hoechst₃₃₃₄₂ dye in the absence (upper panel) or presence (lower panel) of verapamil. All cell lines were analyzed by a cell sorter. <b>B. Expression profiles of stem cell markers in SP cells and MP cells.</b> Expression of stem cell markers (<i>SOX2</i>, <i>POU5F1</i> and <i>NANOG</i>) in SP cells and MP cells derived from LHK2, SW480 and MCF7 cells was determined. <i>GAPDH</i> was used as an internal positive control. <b>C. Quantitative real-time PCR analysis of SOX2 mRNA expression in SP and MP cells.</b> The MP cells were used for the control, which was set as 1.0. Data are expressed as mean + SD of relative values compared to MP cells. <b>D. Expression of </b><b><i>SMCP</i></b><b> in SP and MP cells.</b> Expression of <i>SMCP</i> in SP cells and MP cells derived from LHK2, SW480 and MCF7 cells was determined. <b>E. SMCP is localized in mitochondria.</b> LHK2 cells transfered with GFP-fused SMCP gene were fixed and stained with Mitotracker Red followed by DAPI and then visualized by laser confocal microscopy (Magnification, ×200). Green indicates GFP-fused SMCP. Red indicates mitochondria. Blue indicates nucleus.</p

Expression profiles of SMCP and identification of a novel variant form of SMCP.

<p><b>A. SMCP expression profiles in normal human organs.</b> SMCP mRNA expression in normal tissues (heart, brain, placenta, lung, liver, skeletal muscle, kidney, pancreas, spleen, thymus, prostate, testis, ovary, small intestine, leukocyte, colon, stomach and bone marrow) was investigated by RT-PCR. cDNAs were obtained from TAKARA BIO INC. (Human Multiple Tissue cDNA Panels I and II). <i>GAPDH</i> was used as an internal positive control. <b>B. SMCP expression profiles in human cancer cells.</b> SMCP mRNA expression in lung cancer line cells (Sq-1, Lc817, 86-2, Lu99, 1-87, A549), renal cancer line cells (Caki-1, ACHN, SMKTR1, SMKTR2, SMKTR3, SMKTR4) and pancreas cancer line cell (HPC3) was investigated by RT-PCR. cDNAs were generated by total RNAs derived from cell lines. <i>GAPDH</i> was used as an internal positive control. <b>C. SMCP expression profiles in human lung cancer tissues. </b><i>SMCP</i> mRNA expression in human lung cancer tissues was investigated by RT-PCR. #1 and #2 are adenocarcinoma cases. #3 and #4 are squamous cell carcinoma cases. #5 is a large cell carcinoma case. T indicates lung cancer tissue. N indicates adjacent lung normal tissue. <i>GAPDH</i> was used as an internal positive control. <b>D. Schema of novel variant form of SMCP.</b> SMCP wild type (variant 1) is composed of exon 1 and exon 2. The novel isoform of SMCP (variant 2) has only one exon with a 5′ terminal additional extension. <b>E. Expression profiles of SMCP variant 1 and variant 2 in the testis and cancer cells.</b> SMCP mRNA expression was investigated by RT-PCR using an F1 and F2 mixture primer as a forward primer and R1 primer as a reverse primer in testis and cancer cells. Variant 1 was a 152-bp PCR product, and variant 2 was a 624-bp PCR product. <b>F. Expression of SMCP protein.</b> Detection of SMCP protein in HEK293T cells transfected with expression vectors of variant1 CDS and variant2 as assessed by Western blot analysis with polyclonal SMCP antibody. Beta-actin was used as a protein loading control.</p

SMCP has a role in the tumor-initiating ability in LHK2 lung adenocarcinoma cells.

<p><b>A. Expression of SMCP in SMCP-overexpressed LHK2 cells.</b> Expression of SMCP in LHK2-SMCP cells was confirmed by RT-PCR. <b>B. Representative picture of tumors injected with SMCP-overexpressed LHK2 cells. C. Tumor growth of LHK2-SMCP cells and LHK2-Mock cells.</b> One×10³ and 1×10⁴ of LHK2-SMCP cells and LHK2-Mock cells were injected into NOD/SCID mice subcutaneously. Data are means + SD. Asterisks represents statistical significant difference. Student's t-test. <i>P</i><0.05.</p

Tumor-initiating ability of SP and MP cells.

<p>Incidence indicates the number of tumor formation/number of injections.</p><p>Tumor volumes are mean±S.D.</p

Tumor-initiating ability of SMCP siRNA and Control siRNA.

<p>Incidence indicates the number of tumor formation/number of injections.</p><p>Tumor volumes are mean±S.D.</p>*<p>P<0.01, compared with tumor volumes of 2×10⁵ Control siRNA cells.</p>†<p>P<0.05, compared with tumor volumes of 1×10⁴ Control siRNA cells.</p

Establishment of SP clones and MP clones from the human colon cancer cell SW480.

<p>(A) Summary of establishment of SP clone cells and MP clone cells. SP analysis was performed using SW480 cells. SP cells and MP cells were single cell-sorted using a cell sorter. Single cell platement was confirmed by microscopy. After several weeks of <i>in vitro</i> culture, 11 of 96 wells of MP cells and 12 of 96 wells of SP cells showed cell growth. (B) SP analysis of SP clone cells. SP-A, SP-, SP-C, SP-E and SP-H cells were analyzed for SP phenotypes. Percentage indicate the ratio of SP cells. (C) SP analysis of MP clone cells. MP-B, MP-C, MP-D, MP-E, MP-F, MP-G, MP-H and MP-K cells were analyzed for SP phenotypes. Percentage indicate the ratio of SP cells.</p

SP clones and MP clones showed very stable phenotypes in <i>in vitro</i> culture.

<p>SP-A, SP-B, SP-H, MP-B, MP-D and MP-K cells were analyzed for SP phenotypes. The assay was performed before (left) and after (right) 2 months of <i>in vitro</i> culture. Percentage indicate the ratio of SP cells.</p

Tumor initiating ability in colon cancer cell line.

<p>Tumor initiating ability in colon cancer cell line.</p