Transient silencing of the CDKN2A gene was achieved using a pool of four sirna duplexes

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Sybil Palmer
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1 Supplementary Methods Small interfering RNA (sirna) knockdown of CDKN2A (p16 INK4a /p14 ARF ) Transient silencing of the CDKN2A gene was achieved using a pool of four sirna duplexes that each targeted a sequence shared by the p14 ARF and p16 INK4a transcripts (ON- TARGETplus SMARTpool, Dharmacon, Lafayette, CO). The target sequences were as follows: 5 -GATCATCAGTCACCGAAGG-3, 5 -AAACACCGCTTCTGCCTTT-3, 5 - TAACGTAGATATATGCCTT-3, and 5 -CAGAACCAAAGCTCAAATA-3. A mixture of four nontargeting sirna duplexes was used as a negative control (ON-TARGETplus Nontargeting Pool, Dharmacon). Transfections of IMR-32 and NGP cells were performed using the DharmaFECT 2 lipid transfection reagent (Dharmacon) according to the manufacturer s instructions. The final concentrations of DharmaFECT 2 and the sirna pool in each transfection were 0.2% (v/v) and 100 nm, respectively. The efficiency of CDKN2A knockdown was evaluated by quantitative real-time reverse transcription PCR (qrt-pcr) 24 h after transfection, using a primer pair that measures the transcript levels of both p14 ARF and p16 INK4a (primer sequence available in the RTPrimerDB database, ID #8207, see In cell viability experiments aimed at evaluation of the nutlin-3 response following sirna knockdown of CDKN2A, cells were exposed to CDKN2A sirna or nontargeting negative control sirna for 16 h before nutlin-3 was administered. Lentiviral-mediated short hairpin RNA (shrna) knockdown of CDKN2A Specific knockdown of the two major transcripts of the CDKN2A gene or stable simultaneous silencing of both transcripts was accomplished by shrna constructs directed against previously published target sequences (1): 5 -GAACATGGTGCGCAGGTTC-3 (sequence located in the unique first exon of p14 ARF ), 5 -GAGGAGGTGCGGGCGCTGC-3 (sequence Supplementary Data 1/10

2 located in the unique first exon of p16 INK4a ), and 5 -GGCAGTAACCATGCCCGCA-3 (sequence located in exon 2, which is common to both transcripts). An shrna directed against firefly luciferase was used as a negative control: 5 - GTGCGTTGTTAGTACTAATCCTATTT-3. Lentiviral vector construction, virus production, infection of NGP cells, and puromycin selection were performed as previously described (2). The silencing efficiency was assessed by qrt-pcr using primers specific for p14 ARF (RTPrimerDB ID #3519) or p16 INK4a (#4455). Tetracycline-inducible overexpression of CDKN2A Stable IMR-5/75 and SHEP cell lines overexpressing p14 ARF or p16 INK4a under control of a tetracycline repressor protein were generated using Gateway Technology (Invitrogen, Paisley, UK), as described elsewhere (3), starting from full-length open reading frames of p14 ARF and p16 INK4a that originated from a pool of neuroblastoma cell line cdna. Control clones allowing tetracycline-inducible lacz expression were constructed in parallel. The level of overexpression was measured by qrt-pcr 24 h after addition of 1 µg/ml tetracycline or vehicle control, using primers specific for p14 ARF (RTPrimerDB ID #3519) or p16 INK4a (#4455). In experiments aimed at evaluation of the nutlin-3 response after overexpression of p14 ARF or p16 INK4a, cells were exposed to 1 µg/ml tetracycline or vehicle control for 16 h before nutlin-3 was administered. Statistical analysis Analysis of cell viability data was performed using GraphPad Prism software version 5.01 for Windows (GraphPad Software, San Diego, CA). The nutlin-3 concentrations that caused 50% reduction in cell viability (IC 50 values) were computed by fitting a sigmoidal curve with variable slope to the dose-response data. Differences in IC 50 values between neuroblastoma Supplementary Data 2/10

3 cell lines with different genetic characteristics were analyzed by two-sample t-tests. Differences in genetic characteristics between MYCN-amplified and MYCN-nonamplified neuroblastoma cell lines were analyzed by Mann-Whitney tests. Statistical tests were twosided and were considered significant at P<0.05. For experiments with inducible model systems, IC 50 ratios were calculated as the IC 50 value of nutlin-3 in the presence of the inducing agent (i.e., CDKN2A sirna or tetracycline) divided by the IC 50 value of nutlin-3 in the presence of the appropriate negative control. An IC 50 ratio <1 indicates that the inducing agent potentiates the response to nutlin-3, whereas an IC 50 ratio >1 corresponds to inhibition of the nutlin-3 response. The IC 50 ratios from the individual experiments were then used to compute a mean IC 50 ratio and corresponding 95% confidence interval (CI) at 24, 48, and 72 h of nutlin-3 treatment. Caspase-3 and caspase-7 data from nutlin-3 experiments in the tetracycline-inducible CDKN2A overexpression system were analyzed by calculating the fold change in caspase-3 and caspase-7 activity at a certain nutlin-3 concentration when tetracycline was given compared to when tetracycline was absent. These ratios of caspase-3 and caspase-7 activity from the individual experiments were then used to compute a mean ratio and corresponding 95% CI at each nutlin-3 concentration analyzed. This approach is less suited for analysis of caspase-3 and caspase-7 data from a noninducible experimental model system, because of inherent biological variability between independent cell cultures. Therefore, caspase-3 and caspase-7 data from nutlin-3 experiments in stable shrna knockdown cell lines were analyzed using a quantitative measure that takes the change in caspase activity over multiple nutlin-3 concentrations into account. To this purpose, a sigmoidal curve with variable slope was fit to the log-transformed measurement values of caspase-3 and caspase-7 activity, with the top of the curve set equal to the log-transformed maximum caspase-3 and caspase-7 activity observed in nutlin-3-treated cells with a control shrna construct, and the Supplementary Data 3/10

4 concentration of nutlin-3 that induced a response halfway between the baseline and maximum (EC 50 value) was calculated using GraphPad Prism software version 5.01 for Windows (GraphPad Software). The EC 50 values from the individual experiments were then used to compute a mean EC 50 value and corresponding 95% CI at 24, 48, and 72 h of nutlin-3 treatment. References 1. Voorhoeve PM, Agami R. The tumor-suppressive functions of the human INK4A locus. Cancer Cell 2003;4: Van Maerken T, Speleman F, Vermeulen J, Lambertz I, De Clercq S, De Smet E, et al. Small-molecule MDM2 antagonists as a new therapy concept for neuroblastoma. Cancer Res 2006;66: Henrich KO, Bauer T, Ehemann V, Deubzer H, Gogolin S, Fischer M, Benner A, Schwab M, Westermann F. CAMTA1, a 1p36 tumor suppressor candidate, inhibits growth and activates differentiation programmes in neuroblastoma cells. Submitted for publication. Supplementary Data 4/10

5 Table S1. IC 50 values after nutlin-3 treatment and MDM2, CDKN2A, and MYCN copy number status of neuroblastoma cell lines with wild-type p53 IC 50 value (µm)* Copy number status Cell line IC 50 at 24 h IC 50 at 48 h IC 50 at 72 h MDM2 CDKN2A MYCN CHP N N amp CHP-901 > N N amp CHP-902R N N amp CLB-GA > N N N GICIN N N amp IMR N N amp LA-N N N amp LA-N-6 >32 > N del N NBL-S N N N NGP amp N amp SHEP >32 >32 >32 N del N SH-SY5Y N N N SJNB N N N SJNB-10 > N N amp SK-N-BE(1n) N N amp SK-N-SH N N N SMS-KAN > N N amp SMS-KCNR N N amp STA-NB-1.2 > N N amp STA-NB N del amp STA-NB-8 > N N amp STA-NB N N amp STA-NB N N amp TR-14 > amp N amp UHG-NP > N N amp *Concentration of nutlin-3 resulting in 50% reduction in cell viability at 24, 48, and 72 h of treatment. Copy number status: amp, amplified; del, homozygously deleted; N, nonamplified and nondeleted. IC 50 values were not significantly different between cell lines with and without MDM2 amplification (P>0.05 at 24, 48, and 72 h of treatment). The presence of homozygous CDKN2A deletion was strongly associated with a higher IC 50 value at 48 and 72 h of treatment (P=0.009 and P<0.001, respectively). This association was not yet clear at 24 h of treatment (P>0.05). Amplification of MYCN did not have an impact on the IC 50 values at 24 and 48 h of treatment (P>0.05 at both time points), but was associated with a lower IC 50 value at 72 h of treatment (P=0.023). However, after exclusion of cell lines with homozygous CDKN2A deletion, no differences in IC 50 values were observed with respect to MYCN amplification status (P>0.05 at 24, 48, and 72 h of treatment). Supplementary Data 5/10

6 Table S2. Mutation status of p53 and copy number status of MDM2, CDKN2A, and MYCN of all 34 neuroblastoma cell lines in this study Cell line p53* MDM2 CDKN2A MYCN CHP-134 wt N N amp CHP-901 wt N N amp CHP-902R wt N N amp CLB-GA wt N N N GICIN-1 wt N N amp IMR-32 wt N N amp LA-N-1 mut N N amp LA-N-2 mut N N amp LA-N-5 wt N N amp LA-N-6 wt N del N N-206 mut N N amp NBL-S wt N N N NGP wt amp N amp NLF mut N N amp NMB mut N N amp SHEP wt N del N SH-SY5Y wt N N N SJNB-1 wt N N N SJNB-8 mut N N amp SJNB-10 wt N N amp SK-N-AS mut N N N SK-N-BE(1n) wt N N amp SK-N-BE(2c) mut N N amp SK-N-FI mut N N N SK-N-SH wt N N N SMS-KAN wt N N amp SMS-KCNR wt N N amp STA-NB-1.2 wt N N amp STA-NB-3 wt N del amp STA-NB-8 wt N N amp STA-NB-9 wt N N amp STA-NB-10 wt N N amp TR-14 wt amp N amp UHG-NP wt N N amp *Mutation status: mut, mutated; wt, wild-type. Copy number status: amp, amplified; del, homozygously deleted; N, nonamplified and nondeleted. No significant difference in p53 mutation status nor in MDM2 and CDKN2A copy number status was found between MYCN-amplified and MYCNnonamplified neuroblastoma cell lines (P>0.05 for all three comparisons). Supplementary Data 6/10

7 Figure S1. SK-N-AS cells, of which the p53 gene could not be fully sequenced at the 3 end, were found to have a homozygous deletion of exons 10 and 11 of p53. Shown is the haploid copy number status of exon 5 (left), exon 10 (middle), and exon 11 (right) of p53 in normal human genomic DNA and three human neuroblastoma cell lines, as determined by quantitative real-time PCR (qpcr) with SYBR Green I detection chemistry and qbase PLUS analysis (Biogazelle, Ghent, Belgium). Two reference genes, SDC4 and TNFRSF17, were used for normalization. The haploid copy number level of each p53 exon in normal human genomic DNA was set to 1. Primer sequences are available in the RTPrimerDB database ( exon 5 of p53 (RTPrimerDB ID #1186), exon 10 of p53 (#8208), exon 11 of p53 (#8209), SDC4 (#15), and TNFRSF17 (#14). Bars, SEM of duplicate wells; arrows, homozygous deletion of exons 10 and 11 of p53 in SK-N-AS cells. Supplementary Data 7/10

8 Figure S2. SK-N-SH and SH-SY5Y cells have a normal CDKN2A gene copy number, whereas this gene is homozygously deleted in SHEP cells. Copy number levels of CDKN2A in genomic DNA isolated from these neuroblastoma cell lines and in normal human genomic DNA were determined by SYBR Green I qpcr with qbase PLUS analysis (Biogazelle). Two reference genes, SDC4 and TNFRSF17, were used for normalization. The haploid copy number level of CDKN2A in normal human genomic DNA was set to 1. Primer sequences are available in the RTPrimerDB database ( CDKN2A (RTPrimerDB ID #8210), SDC4 (#15), and TNFRSF17 (#14). Bars, SEM of duplicate wells. Supplementary Data 8/10

9 Figure S3. Overexpression of p14 ARF or p16 INK4a does not affect the sensitivity of SHEP cells to nutlin-3. A, qrt-pcr analysis of p14 ARF and p16 INK4a expression in SHEP cells stably transfected with a tetracycline-inducible expression vector for a negative control construct (SHEP-Tet-lacZ), p14 ARF (two independent clones, SHEP-Tet-p14.1 and SHEP-Tet-p14.2), or p16 INK4a (two independent clones, SHEP-Tet-p16.1 and SHEP-Tet-p16.2). Cells were treated with 1 µg/ml tetracycline or vehicle control for 24 h. Bars, SEM of duplicate wells. B, effect of p14 ARF and p16 INK4a overexpression on the viability of SHEP cells after treatment with nutlin-3. Cells were treated with 1 µg/ml tetracycline or vehicle control and subsequently exposed to nutlin-3 for 24 h, followed by cell viability analysis. Induction of p14 ARF or p16 INK4a expression did not increase the sensitivity of SHEP cells to nutlin-3. Similar findings were observed after 48 and 72 h of nutlin-3 treatment and in an independent replication of the experiment. Bars, SD of triplicate wells. RLU, relative luminescence units. Supplementary Data 9/10

10 Supplementary Data 10/10

Supplementary Figure 1. NORAD expression in mouse (A) and dog (B). The black boxes indicate the position of the regions alignable to the 12 repeat

Supplementary Figure 1. NORAD expression in mouse (A) and dog (B). The black boxes indicate the position of the regions alignable to the 12 repeat units in the human genome. Annotated transposable elements