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Blood, Vol. 113, Issue 14, 3172-3181, April 2, 2009
Functional involvement of RINF, retinoid-inducible nuclear factor (CXXC5), in normal and tumoral human myelopoiesis
Blood Pendino et al.
113: 3172
Supplemental materials for: Pendino et al
Files in this Data Supplement:
- Document 1. Supplemental materials and methods (PDF, 53.3 KB)
- Table S1. Microarray results and selection process to identify early target genes of retinoic acid in NB4 cells (PDF, 29 KB)
- Table S2. List of other genes identified as early induced by retinoic acid in NB4 cells (PDF, 25.3 KB)
- Figure S1. Expression of RINF protein in total extracts from NB4, NB4-LR1, and NB4-LR2 cells treated with ATRA (1 µM) (JPG, 25.5 KB)
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RINF was detected with our customized polyclonal rabbit antibody (see Methods) that reveals a specific band at 33 kDa. ACTIN was used as a loading control. RINF expression was more pronounced in NB4 cells, than in the two resistant subclones NB4-LR1 and NB4-LR2.
- Figure S2. CXXC5 mRNA expression in blasts derived from three (PML-RARa positive) APL patients (JPG, 49.3 KB)
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Cells were treated or not with ATRA (1 µM) during 4h. Data have been extracted from the publically available database Arrayexpress (E-MEXP-149). Microarray experiments were performed by Meani et al.27 according to Affymetrix GeneChip Human Genome HG-U133A and HG-U133B (2 represented hybridizations for each sample). The quantitation type used for the expression value measurement is affymetrix:CHPSignal. The 3 probes targeting CXXC5 expression were probe a (222996_s_at), b (224516_s_at), and c (233955_x_at). The two red dotted lines indicate untreated and ATRA-treated group means. We applied the Paired-student’s t-Test to show that the values for these two groups were significantly different (p-value < 0.05).
- Figure S3. shRNA-mediated silencing of RINF delays ATRA-induced terminal differentiation of HL60 cells (JPG, 99.4 KB)
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HL60 cells were infected with Empty, shRNA/Scramble, shRNA/RINF-3 or shRNA/RINF-4 lentiviral vectors and selected. Cells were then treated or not with ATRA (1µM). RINF mRNA expression was assessed by quantitative RT-PCR (% of untreated mock-control at 6 hours of culture). Terminal differentiation was assessed by the NBT reduction assay at day 2 and by cell morphology at day 6 and (scale bars, 25 µm).
- Figure S4. RINF over-expression is not sufficient to induce differentiation of NB4 and HL60 cell lines (JPG, 64.8 KB)
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(A) A retroviral system derived from Murine Stem Cell Virus (MSCV) was used to over-express RINF in the two cell lines. The cells were infected and sorted 9 days post-infection for GFP expression. (B) For the two cell lines, RINF mRNA expression was measured (here at day 10) by quantitative RT-PCR and represented in % of their respective mock control (+∕− s.e.m). (C ) Cells were cytospined and stained with MGG for cell morphology analysis here visualized at two different magnitudes (scale bars, 25 µm). No sign of differentiation was noticed even after 10 days of RINF over-expression. A few percentage of cell death (about 10% of the total) was consistently noticed in cell cultures over-expressing RINF.
- Figure S5. CXXC5 mRNA expression in CD34+ cells derived from 55 patients suffering from myelodysplasia (MDS) (JPG, 46.3 KB)
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Data have been extracted from the publicly available databases Arrayexpress (E-GEO-4619) and Gene Expression Omnibus (GDS2118). Microarray experiments were performed by Pellagatti et al.46 according to GeneChip Human Genome U133 Plus 2.0 arrays (Affymetrix). Each point represents 1 patient with MDS or a healthy individual. The three red dotted lines indicate group means (998.1, 512.2, and 985.9) for normal healthy donors (n=11), MDS with deletion 5q (n=20) or MDS without del 5q (n=35). The values for Normal and MDS with del(5q) were significantly different according to the Wilcoxon two sample test (p<0.003). URLs: http://www.ebi.ac.uk/arrayexpress/ (ArrayExpress) and http://www.ncbi.nlm.nih.gov/geo/ (Gene Expression Omnibus).
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