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, Biomarker Discovery, Western Blot, Control, Concentration Assay, Quantitation Assay, Fractionation, Knock-Out, CRISPR, Double Knockout, Microscopy

Journal: Journal of Biological Chemistry

Article Title: Targeting O-Glycosyltransferase (OGT) to Promote Healing of Diabetic Skin Wounds

doi: 10.1074/jbc.m113.513952

Figure Lengend Snippet: FIGURE 1. Hyperglycemia increases O-GlcNAc modification and retards wound healing in human keratinocytes. HaCaT cells were cultured in media supplemented with glucose to the final concentrations indicated. A, representative immunoblot (IB) of cell lysates separated by SDS-PAGE and immunoprobed with antibodies to (i) RL2, which recognizes the O-GlcNAc modification, and (ii) GAPDH as a loading control. B, the RL2 signal was quan- tified relative to the GAPDH loading control (n 3). C and D, scratch assays. Human keratinocyte monolayers were incubated in DMEM with the indicated glucose concentrations. Cells were scratched with a pipette tip, and micro- graphs were made at 0 and 16 h. Wound sizes were then measured using image analysis software. Representative micrographs are shown in C and quantified in D. n 11 for all conditions. Error bars reflect the S.E. * indicates p value 0.07 and ** indicates p value 0.0005 as compared with normal glucose levels (5.5 mM). The p value between 25 and 100 mM is 0.01.

Article Snippet: Rabbit monoclonal antibodies to GAPDH were from Cell Signaling (Danvers, MA).

Techniques: Modification, Cell Culture, Western Blot, SDS Page, Control, Incubation, Transferring, Software

Journal: Journal of Biological Chemistry

Article Title: Targeting O-Glycosyltransferase (OGT) to Promote Healing of Diabetic Skin Wounds

doi: 10.1074/jbc.m113.513952

Figure Lengend Snippet: FIGURE 3. OGT knockdown using siRNA accelerates wound healing. HaCaT cells were transfected with 100 nM siRNA against OGT or a scrambled control siRNA. A and B, cell lysates of confluent cultures were subjected to immunoblot (IB) with antibodies to (i) O-GlcNAc (RL2), (ii) OGT, and (iii) GAPDH (control) (A) and quantified (B). Untr, untreated cells. C and D, 60 h after transfection with siRNAs, the confluent cultures were scratched, and micrographs were obtained at 0, 16, and 26 h (C). The open wound area was quantified using image analysis software (D). n 7 for the untransfected cells, n 7 for control siRNA, and n 6 for OGT siRNA. Error bars reflect the S.E. * indicates p value 0.05 as compared with controls.

Article Snippet: Rabbit monoclonal antibodies to GAPDH were from Cell Signaling (Danvers, MA).

Techniques: Knockdown, Transfection, Control, Western Blot, Software