Table 1.
Primary and secondary antibodies.
Type: Mono, monoclonal; Poly, polyclonal. Host: Rb, rabbit; Mo, mouse; Ho, horse; Go, goat. Dilution (for methods): WB, Western blotting; IHC, immunohistochemistry. Localization of the antigen in positive controls: N, nuclear; C, cytoplasmic.
Table 2.
F, forward primer; R, reverse primer.
Primers.
Fig 1.
Representative IHC analysis of putative biomarkers in tumor samples.
Representative expressions of DDX39A, HMGA1, HMGA2, HOXC9, and PBX1 in the tumor samples No. 2 (corresponding to the NBL-13 retinoid-sensitive cell line) and No. 18 (corresponding to the NBL-36 retinoid-resistant cell line). Representative expressions of these markers in positive control samples are also given for comparison. Original magnification, 200×.
Fig 2.
Analysis of endogenous gene expressions of putative biomarkers in patient-derived NBL cell lines.
Representative gel electrophoresis images captured using a UV transilluminator are shown for each sample, including GAPDH as a reference housekeeping gene.
Fig 3.
Analysis of endogenous protein levels of putative biomarkers in patient-derived NBL cell lines.
Representative immunoblot images captured using X-ray film are shown for each sample. Since looking for reliable loading control protein that would provide satisfactory results throughout all analyzed NBL cell lines was showed to be difficult, loaded amounts of proteins for each sample are documented on the corresponding PVDF membrane scan. As the NBL-30 cell lysate was not obtained in the appropriate quality, this cell line was finally excluded from this screening.
Table 3.
Clinical description of the patients included in this study.
Age at the time of biopsy (in months). Tumor histology according Shimada system: UH, unfavorable histology; FH, favorable histology; N/A, not available. INSS stage according to the International Neuroblastoma Staging System Committee (INSS) system. NBL risk category: LR, low risk; IR, intermediate risk; HR, high risk. Status: NED, no evidence of disease; DOD, dead of disease; AWD, alive with disease.
Table 4.
Results of IHC analyses of DDX39A, HMGA1, HMGA2, HOXC9, and PBX1 expression in tumor samples.
The percentage of antigen-positive tumor cells (TC) was counted and categorized into five levels: - (0% positive TC), +/- (1-10% positive TC), + (11-50% positive TC), ++ (51–80% positive TC), and +++ (81–100% positive TC). The intensity of immunostaining (immunoreactivity, IR) was classified as none (0), weak (1), medium (2), or strong (3). PQ, poor quality of the specimen (impossible to perform a correct evaluation for the respective biomarker). Sample No. 13 was not available for this IHC analysis, sample No. 14 was excluded due to its overall poor quality.
Table 5.
Cell viability was measured after the treatment with the respective retinoid compared to untreated cells. Green/red colors indicate more sensitive/resistant cell lines that were selected for the next part of this study. PQ - poor quality, * - statistical significance P < 0.05, ** - statistical significance P < 0.01. NBL-15 cell line was finally excluded from the evaluation due to overall reduced proliferation activity.
Table 6.
An overview of detected changes in putative marker expression in sensitive and resistant NBL cell lines after treatment with natural or synthetic retinoids.
The upward arrow indicates an increase in expression of the respective mRNA and/or protein, and the downward arrow indicates a decrease in mRNA and/or protein expression.
Fig 4.
Analysis of candidate biomarkers mRNA expression.
DDX39A (A), HMGA1 (B), HMGA2 (C), HOXC9 (D), and PBX1 (E). Relative gene expression was determined in 6 patient-derived and 2 reference cell lines after 7 days of treatment with natural (ATRA, 9-cis-RA, 13-cis-RA) and synthetic (BEX, 4 HPR) retinoids administered at a 1 μM concentration. Experiments were repeated in triplicate. Representative gel electrophoresis images captured using a UV transilluminator are shown for each sample, including HSP90AB1 as a reference gene.
Fig 5.
Analysis of candidate biomarkers protein expression.
(A) DDX39A–49 kDa; (B) HMGA1–18 kDa; (C) HMGA2–18 kDa; (D) HOXC9–29 kDa; and (E) PBX1–46 kDa. Relative protein expression was determined in 6 patient-derived and 2 established NBL cell lines after treatment with natural (ATRA, 9-cis-RA, 13-cis-RA) and synthetic (BEX, 4-HPR) retinoids administered at a 1 μM concentration for 7 days. Images of the representative immunoblot captured using X-ray film are shown for each sample, including GAPDH as a reference protein. Experiments were repeated in triplicate.