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

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 ELISA plates.

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% Tween, treated with goat anti-rabbit IgG.

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 450 nm in a plate reader.

#### **2.8 Caspase 3 activity**

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

**103**

*Targeting MYC and HDAC8 with a Combination of siRNAs Inhibits Neuroblastoma Cells…*

CASP3-C). 50ug protein was mixed with the peptide substrate, Ac-DEVD-pNA (p-nitroanilide), in the presence of 10 mM DTT. Caspase 3 hydrolyzes the substrate, releasing p-nitroaniline, which is read at 405 nm. The specificity of caspase 3

HepG2 cells were used for miR-665 target validation, because miR-665 does not inhibit the growth of these cells. When mouse neuroblastoma cells were used for target validation, the negative control luciferase vector plasmid without any target 3'-UTR showed a 50% decrease in luciferase activity when co-transfected with miR-665 compared to negative control miRNA. This decrease in luciferase activity was non-specific and was caused by cell growth inhibition due to miR-665 transfection. To validate miR-665 targets, HepG2 cells were grown for 24 h in a 96-well plate. Cells were then co transfected with 100 ng luciferase expression plasmids containing the 3'-UTR for HDAC8, c-MYC, or MYCN, or the empty vector without any target 3'UTR, plus 100 nM negative control miRNA or miR-665 with Dharmafect Duo transfection agent. After 48 h of cotransfection, luciferase activity was measured using the Active Motifs LightSwitch luciferase assay kit. Luminescence was

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

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

When tumors reached 100 mm3 in size, mice were divided into two groups with

activity was determined in the presence of the inhibitor, Ac-DEVD- CHO.

**2.9 Target validation using luciferase expression plasmids**

read on a Molecular Devices Soft Max Pro5 luminometer.

2 N H2SO4. Samples were read at 450 nm in a plate reader.

**2.11 Neuroblastoma tumor model**

8–10 mice in each group.

Antisense Sequence:

seen and tumors were measured with a caliper.

siRNA-HDAC8 (S88696), Sense Sequence: (5′----3′).

siRNA-MYC (S70224), Sense Sequence: (5′------3′).

Intratumoral delivery of siRNA.

CGACGGAAAUUUGACCGUAtt.

UACGGUCAAAUUUCCGUCGca.

AGGUAGUGAUCCUCAAAAAtt.

**2.10 Histone acetylation**

*DOI: http://dx.doi.org/10.5772/intechopen.96021*

CASP3-C). 50ug protein was mixed with the peptide substrate, Ac-DEVD-pNA (p-nitroanilide), in the presence of 10 mM DTT. Caspase 3 hydrolyzes the substrate, releasing p-nitroaniline, which is read at 405 nm. The specificity of caspase 3 activity was determined in the presence of the inhibitor, Ac-DEVD- CHO.
