**2.10 Histone acetylation**

*Pheochromocytoma, Paraganglioma and Neuroblastoma*

Assay as per the manufacturer's instructions.

**2.5 Quantitation of miR-665 in transfected cells**

software v.6.0 and the 2∆∆Ct method.

product was read at 405 nm in a plate reader.

Tween, treated with goat anti-rabbit IgG.

**2.7 HDAC8 and c-MYC protein quantitaion via ELISA**

**2.6 Total HDAC activity**

c-MYC levels via ELISA.

MD, USA).

ELISA plates.

450 nm in a plate reader.

**2.8 Caspase 3 activity**

negative control miRNA, miR-665, negative control siRNA, c-MYC siRNA, or HDAC8 siRNA plus Lipofectamine RNAimax. Transfected cells were plated in T25 flasks. After 48–72 h, cell extracts were prepared in assay buffer as described by Khandelia, *et al.* [16]. Assay buffer consisted of 20 mM Tris–HCL pH 7.5, 150 mM NaCl, 5 mM EDTA, 10% glycerol, 1% Nonidet P40, and protease inhibitor cocktail from Sigma (P8340). Protein concentrations were determined using Pierce's BCA

miR-665 inhibit cell growth compared to untreated cells and cells treated with negative control miRNA. Assays were normalized using equal concentrations of protein (50–100 ug) from untreated, negative control miRNA-, and miR-665 treated cells in assessing total HDAC and Caspase 3 activity, and HDAC8 and

Mouse neuroblastoma cells were transfected with 100 nM miR-665 mimic and negative control cel-miR-67. 48 h post-transfection, total RNA was extracted from three biological replicates per treatment using the Qiagen RNEasy mini kit. miR-665 was quantitated via realtime qPCR by Arraystar, Inc. (Rockville,

Real-time PCR was performed for each RNA sample to quantify miR-665 and the housekeeping gene, U6. According to the standard curve, mRNA concentrations in each sample are determined directly using Rotor-Gene Real-Time Analysis

Total HDAC activity was measured in 50–75ug of protein from cell extracts prepared from untreated, or negative control miRNA- or miR 665-transfected cells using the Biovision kit (#K331–100). Acetylated HDAC substrate and other reagents were added according to the manufacturer's instructions and the final deacetylated

HDAC8 and c-MYC proteins were quantitated using cell extracts prepared from untreated or 1 mM Bt2cAMP treated cells, or negative control miRNA- or miR-665- transfected cells via ELISA. 100ug protein per sample was mixed with 0.02 M carbonate coating buffer (pH 9.5) and added to 96-well BD-Falcon

Samples were incubated at 4°C for 15 h. Wells were blocked with 10% FBS in PBS, treated with antibodies (diluted 1:30) specific for HDAC8 (SC11405) or c-MYC (SC-798), and incubated at 37°C for 2 h. Samples were washed with PBS + 0.05%

HRP secondary antibody (diluted 1:500), and incubated at 37°C for 1 h. Wells were washed and treated with substrate TMB and incubated at room temperature for 30 min, and then the reaction was stopped with 2 N H2SO4. Samples were read at

Caspase 3 activity was measured in 50ug protein from untreated, Bt2cAMPtreated, or miR-665-transfected cells using Sigma Aldrich's colorimeter kit (Code

**102**

Histone acetylation was quantified via ELISA in cell extracts prepared from cells transfected with negative control miRNA, miR-665, negative control siRNA, or HDAC8 siRNA. 100ug protein was mixed with 0.02 M carbonate coating buffer (pH 9.5), added to 96-well BD Falcon ELISA plates, and incubated at 4°C for 15 h. Wells were blocked with 10% FBS in PBS, treated with acetylated antibodies (diluted 1:30) for Ac H2B (Lys 5/12/15/20), Ac-H3 (lys9), or Ac-H4 (lys16), and incubated at 37°C for 2 h. Samples were washed with PBS + 0.05% Tween, treated with an appropriate HRP-conjugated secondary antibody (diluted 1:500), and incubated at 37°C for 1 h. Wells were washed and treated with substrate TMB and incubated at room temperature for 30 min, and then the reaction was stopped using 2 N H2SO4. Samples were read at 450 nm in a plate reader.

## **2.11 Neuroblastoma tumor model**

Mice experiments were performed with the approval of the institutional Animal Care and Use Committee, IACUC at Nanospectra Biosciences Inc. Houston, Texas.

A/J female mice six weeks old were purchased from Jackson Laboratory, Bar Harbor, Maine, USA. Murine neuroblastoma cells, 1x106 cells in DMEM media with 50% matrigel in 100 ul without fetal bovine serum and without antibiotics were subcutaneously injected on the right flanks. After 12 days, tumor growth can be seen and tumors were measured with a caliper.

When tumors reached 100 mm3 in size, mice were divided into two groups with 8–10 mice in each group.

Intratumoral delivery of siRNA. siRNA-HDAC8 (S88696), Sense Sequence: (5′----3′). CGACGGAAAUUUGACCGUAtt. Antisense Sequence: UACGGUCAAAUUUCCGUCGca. siRNA-MYC (S70224), Sense Sequence: (5′------3′). AGGUAGUGAUCCUCAAAAAtt.

Antisense Sequence: UUUUUGAGGAUCACUACCUtg.

Negative control #2 siRNA (#4390846), and Lipofectamine RNAi Max (#13778075) were purchased from Life Technologies/Ambion.

A total of 3 nmol Negative control siRNA or 3 nmol combinations of siRNA-MYC + siRNA-HDAC8 were mixed with Lipofectamine RNAi max (Liposome) in DMEM media without fetal bovine serum and without antibiotic. siRNA complexed with Lipofectamine in a volume of 30 ul was delivered into tumors by intratumoral injection every third day. Tumors were measured every second day with a caliper and mice were weighed every third day. Tumor volume was calculator with a formula, V = Length X width2/2. Experiment was stopped when the control tumors treated with negative control siRNA reached a tumor burden volume of 1200 mm3. Mice were euthanized by CO2 inhalation 2 days after last treatment with siRNA. Tumors were removed and weighed. Tumors were frozen in liquid nitrogen and stored at -80o C freezers until used for preparation of tumor extracts for ELISA.
