Fig 1.
A. Data from the Human Protein Atlas showing IHC stain intensity for ILEI in melanoma vs breast cancer [69]. B. Data from NCBI GEO database [72] comparing ILEI mRNA levels in primary vs metastatic melanoma patient samples, accession GSE8401, Nprimary = 31, Nmetastatic = 52, meanprimary = 136, meanmetastatic = 323, SDprimary = 63, SDmetastatic = 208, p = 5 x 10−6 by unpaired Student’s t-test [72]. C. Semi-quantitative RT-PCR analysis of ILEI and DCT in primary epidermal melanocytes, WM983, and 1205Lu cells.
Fig 2.
ILEI expression in melanoma cell lines.
A. Immunoblot and semi-quantitative RT-PCR analysis of ILEI and MITF levels in WM3912, WM983B, 501-Mel, Sk-Mel-28, WM793, 1205Lu, and WM9 cells. IB WCL indicates ILEI in the whole cell lysate, whereas IB CM indicates ILEI in protein that was precipitated out of serum-free conditioned medium. B. Quantitative RT-PCR analysis of MITF and ILEI levels in WM3912, 501-Mel, 1205Lu, and WM9 cells. Grey bars indicate MITF mRNA and black bars indicate ILEI mRNA. N = 3, mean +/- SD, transcript levels normalized to GAPDH. C. ILEI ELISA of conditioned medium from WM3912, WM983B, 501-Mel, Sk-Mel-28, WM793, and 1205Lu cells. Prior to conditioned medium analysis, the cells were cultured for 24h in serum-free RPMI. D. Melanoma patient RNA-seq data from cBioPortal [70, 73] showing MITF mRNA z-score vs FAM3C mRNA z-score in melanoma patient samples. The correlation was calculated using Pearson’s correlation coefficient: r = -0.217, N = 471, p = 1.94 x 10−6.
Fig 3.
The effect of phenotype switching on ILEI expression.
A. Immunoblot analysis of MITF and ILEI levels in ILEI-low Sk-Mel-28 cells treated with TGF-β (0 to 48 h, 5 ng/ml). B. Semi-quantitative RT-PCR analysis of MITF, DCT, ILEI, ZEB1, ZEB2, and ACTB levels in ILEI-high WM9 cells treated with vemurafenib (BRAFi, 0 to 48 h, 1 μM). C. Immunoblot analysis of MITF and ILEI levels in ILEI-low 501-Mel stably expressing scrambled shRNA or three independent shRNA hairpins specific for MITF. D. Immunoblot and semi-quantitative RT-PCR analysis of MITF, ILEI, and DCT levels in WM9 stably expressing empty vector or MITF. E. Quantitative RT-PCR analysis of MITF and ILEI levels in 1205Lu cells transiently expressing empty vector or MITF. N = 3, mean +/- SD, mRNA levels normalized to GAPDH, * indicates p < 0.05 by unpaired Student’s t-test. F. Semi-quantitative RT-PCR analysis of MITF and ILEI levels in WM9 and 1205Lu cells stably expressing scrambled shRNA or shRNA targeting ILEI.
Fig 4.
The effect of ILEI knockdown in ILEI-high/MITF-low cells on invasion and proliferation.
A. 3-D spheroid invasion assay of ILEI-low WM983B or ILEI-high WM9 cells in extracellular matrix. B. Transwell migration assay of ILEI-low 501-Mel or ILEI-high WM9 cells. Images are pseudo colored blue. C. Wound healing assay of 1205Lu or WM9 expressing shSCR, shILEI 3, or shILEI 5. Images shown are representative of three independently seeded replicates. D. Quantification of panel E using ImageJ software. N = 3, mean +/- SEM, and * indicates p < 0.05 by unpaired Student’s t-test. Solid bars indicate shSCR, grey bars indicate shILEI 3, and white bars indicate shILEI 5. E. Transwell migration assay of 1205Lu or WM9 expressing shSCR, shILEI 3, or shILEI 5. N = 3, mean +/- SEM, and * indicates p < 0.05 by unpaired Student’s t-test. F. Transwell invasion assay of 1205Lu expressing shSCR, shILEI 3, or shILEI 5. N = 3, mean +/- SEM, and * indicates p < 0.05 by unpaired Student’s t-test. Images are representative of three independently seeded replicates, and pseudo colored in blue. G. Immunoblot analysis of ILEI-high 1205Lu or WM9 expressing shSCR, shILEI 3, or shILEI 5. H. Cell counts of 1205Lu or WM9 expressing shSCR or shILEI 3. Solid lines indicate shSCR and dotted lines indicate shILEI 3. Data is representative of three independently seeded replicates.
Fig 5.
The effect of ILEI knockdown in ILEI-high/MITF-low cells on gene expression.
A. Microarray analysis of ILEI-high 1205Lu or WM9 stably expressing shSCR, shILEI 3, shILEI 4, or shILEI 5. Venn diagrams represent numbers of genes up (1.2-fold) or down (0.8-fold) in shSCR vs shILEI (p < 0.05 in shSCR vs shILEI sample by unpaired Student’s t-test, N = 3). B. Heat map of top 10 genes up or down in shSCR vs shILEI (p < 0.05 in shSCR vs shILEI by unpaired Student’s t-test, N = 3). Heat map was generated using ClustVis [68]. C. Gene set enrichment analysis [66, 67] of genes up in shSCR vs shILEI (>1.2-fold and p < 0.05; 137 genes). Enriched hallmark pathways and transcription factor motifs are shown.
Table 1.
Genes up in shSCR vs shILEI.
Table 2.
Genes down in shSCR vs shILEI.