Journal: Cell Death & Disease

Article Title: MYC transcription activation mediated by OCT4 as a mechanism of resistance to 13- cis RA-mediated differentiation in neuroblastoma

doi: 10.1038/s41419-020-2563-4

Figure Lengend Snippet: a , b Protein expression of c-MYC and MYCN in patient-derived neuroblastoma cell lines established from clinical samples collected at diagnosis (Dx) ( a ) and progressive disease (PD) ( b ). CHLA-20 and SK-N-BE(2) were used as the positive controls for c-MYC and MYCN. The results were replicated in independent experiments. c Dot plots quantitating immunoblots shown in a and b . The values obtained by densitometry were normalized in two ways by c-MYC in CHLA-20 and MYCN in SK-N-BE(2) per blot, and then by GAPDH for loading. d Morphology of SMS-LHN (LHN) cells that were treated with vehicle control or 5 μM 13- cis RA for 14 days and SMS-LHN-R (LHN-R, selected for resistance to 13- cis RA) cells. Cells were treated with 5 μM 13- cis RA (clinically achievable concentration) for 14 days. The observation was replicated in repeat experiments. Scale bar: 200 μM. e Cell cycle analyses of LHN and LHN-R cells treated with vehicle control or 5 μM 13- cis RA for 14 days ( p < 0.01 for LHN, p = 0.07 for LHN-R). Triplicate samples of 10,000 events were analyzed. The error bars represent the SD. Left: representative histograms, right: percentage of cells in each phase of cell cycle. f Basal mRNA levels of MYC and MYCN in LHN and LHN-R cells. Relative quantitation (2 −ΔΔCT ) was used for the analyses of mRNA expression. In LHN-R relative to LHN, MYCN expression was significantly decreased while MYC expression was increased ( p < 0.05) in three independent samples. Symbols: data, Bars: mean. g Expression of c-MYC and MYCN in subcellular fractions (C: cytosolic, N: nuclear) of LHN and LHN-R cells. LDH (cytosolic protein) and Lamin A/C (nuclear protein) were used as fractionation quality controls. The results were reproducible in a repeat experiment. h Effect of MYC knockout (KO) using CRISPR/Cas9 on Cyclin A, a downstream target of c-MYC in LHN-R cells. KO of MYC in both DNA strands was lethal to LHN-R cells, and thus the experiments were conducted in single KO cells. Morphological changes of MYC KO cells is shown in Supplementary Fig. . The results were reproducible in a repeat experiment. i MYC knockout (KO) using a CRISPR/Cas9 system in LHN-R cells. MYC double knockout was lethal to LHN-R cells, and thus the experiments were conducted in MYC single knockout cells. The cells expressing wild-type MYC and MYC KO were treated with 13- cis RA at 5 mM for 14 days, and then the neurite outgrowth was observed under the bright-field microscope. Scale bar: 200 μM.

Article Snippet: TaqMan qPCR primers used in the study are: MYC , Hs02602824_cn (amplicon length 87 bp); MYCN , Hs02718426_cn (amplicon length 81 bp); and GAPDH (Hs.PT.39a.22214836, Integrated DNA Technologies).

Techniques: Expressing, Derivative Assay, Western Blot, Concentration Assay, Quantitation Assay, Fractionation, Knock-Out, CRISPR, Double Knockout, Microscopy