Journal: PLoS ONE

Article Title: Ectopically Expressed Variant Form of Sperm Mitochondria-Associated Cysteine-Rich Protein Augments Tumorigenicity of the Stem Cell Population of Lung Adenocarcinoma Cells

doi: 10.1371/journal.pone.0069095

Figure Lengend Snippet: A. SMCP expression profiles in normal human organs. 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). GAPDH was used as an internal positive control. B. SMCP expression profiles in human cancer cells. 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. GAPDH was used as an internal positive control. C. SMCP expression profiles in human lung cancer tissues. SMCP 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. GAPDH was used as an internal positive control. D. Schema of novel variant form of SMCP. 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. E. Expression profiles of SMCP variant 1 and variant 2 in the testis and cancer cells. 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. F. Expression of SMCP protein. 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.

Article Snippet: Human breast adenocarcinoma cell line MCF7, human lung adenocarcinoma cell line A549 and human embryonal kidney cell line HEK293T were purchased from ATCC (Manassas, VA, USA).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Positive Control, Generated, Derivative Assay, Variant Assay, Transfection, Western Blot