Journal: Molecular Oncology

Article Title: EBP2, a novel NPM‐ALK‐interacting protein in the nucleolus, contributes to the proliferation of ALCL cells by regulating tumor suppressor p53

doi: 10.1002/1878-0261.12822

Figure Lengend Snippet: Knockdown of EBP2 enhances phosphorylation of Akt at S473 in Ba/F3 cells expressing NPM‐ALK. Control Ba/F3 cells (−), Ba/F3‐NPM‐ALK, and Ba/F3‐K210R were transfected with control scrambled siRNA and two kinds of siRNA targeting EBP2 (EBP2 siRNA #1 or EBP2 siRNA #2). Twenty hours after transfection, whole‐cell lysates were prepared and immunoblotted with an anti‐Flag, anti‐phospho‐STAT3 (Tyr705), anti‐STAT3, anti‐phospho‐Akt (Thr308), anti‐phospho‐Akt (Ser473), anti‐Akt, or anti‐β‐actin antibody. The relative phosphorylation levels of STAT3 and Akt are shown in the graphs. Results represent the mean ± SD of three independent experiments. * P < 0.05, *** P < 0.001 significantly different from the control group of Ba/F3 cells transfected with scrambled siRNA. # P < 0.05, ## P < 0.01 significantly different from the group of Ba/F3‐NPM‐ALK transfected with scrambled siRNA.

Article Snippet: Transduced Ba/F3 cells and Ki‐JK cells were transfected with siRNA by electroporation using the Neon transfection system (WAKENYAKU Co., Ltd., Kyoto, Japan) according to the manufacturer's instructions.

Techniques: Knockdown, Phospho-proteomics, Expressing, Control, Transfection

Journal: Molecular Oncology

Article Title: EBP2, a novel NPM‐ALK‐interacting protein in the nucleolus, contributes to the proliferation of ALCL cells by regulating tumor suppressor p53

doi: 10.1002/1878-0261.12822

Figure Lengend Snippet: Knockdown of EBP2 activates p53 through Akt in Ba/F3 cells expressing NPM‐ALK. Ba/F3‐NPM‐ALK was transfected with control scrambled siRNA and two kinds of siRNA targeting EBP2 (EBP2 siRNA #1 or EBP2 siRNA #2). Fourteen hours after transfection, cells were treated with GDC‐0068 (2.5 and 5 μ m ) for 6 h. (A) Whole‐cell lysates were immunoblotted with an anti‐phospho‐Akt (Ser473), anti‐Akt, anti‐phospho‐p53 (Ser15), anti‐p53, anti‐p21, anti‐EBP2, or anti‐β‐actin antibody. The relative phosphorylation or expression levels of Akt, p53, p21, and EBP2 are shown in the graphs. Results represent the mean ± SD of three independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001 significantly different from the control group of Ba/F3‐NPM‐ALK transfected with scrambled siRNA. # P < 0.05, ## P < 0.01, ### P < 0.001 significantly different from the group of Ba/F3‐NPM‐ALK transfected with EBP2 siRNA #1 or EBP2 siRNA #2. (B) Total RNA was prepared and the expression of p21 mRNA was analyzed by quantitative real‐time PCR ( n = 3). Rpl13a mRNA was analyzed as an internal control. Error bars represent the SD of the mean. * P < 0.05; *** P < 0.001 significantly different from the group of Ba/F3‐NPM‐ALK transfected with scrambled siRNA. # P < 0.05 significantly different from the group of Ba/F3‐NPM‐ALK transfected with EBP2 siRNA #1 or EBP2 siRNA #2.

Article Snippet: Transduced Ba/F3 cells and Ki‐JK cells were transfected with siRNA by electroporation using the Neon transfection system (WAKENYAKU Co., Ltd., Kyoto, Japan) according to the manufacturer's instructions.

Techniques: Knockdown, Expressing, Transfection, Control, Phospho-proteomics, Real-time Polymerase Chain Reaction

Journal: Molecular Oncology

Article Title: EBP2, a novel NPM‐ALK‐interacting protein in the nucleolus, contributes to the proliferation of ALCL cells by regulating tumor suppressor p53

doi: 10.1002/1878-0261.12822

Figure Lengend Snippet: The EBP2 knockdown‐induced p53 activation is inhibited by Rapamycin in Ba/F3 cells expressing NPM‐ALK. Ba/F3‐NPM‐ALK was transfected with control scrambled siRNA and two kinds of siRNA targeting EBP2 (EBP2 siRNA #1 or EBP2 siRNA #2). Fourteen hours after transfection, cells were treated with rapamycin (1.25 and 2.5 n m ) for 6 h. (A) Whole‐cell lysates were immunoblotted with an anti‐phospho‐p70 S6 kinase (Thr389), anti‐p70 S6 kinas, anti‐phospho‐Akt (Ser473), anti‐Akt, anti‐phospho‐p53 (Ser15), anti‐p53, anti‐p21, anti‐EBP2, or anti‐β‐actin antibody. The relative phosphorylation or expression levels of p70 S6K, Akt, p53, p21, and EBP2 are shown in the graphs. Results represent the mean ± SD of three independent experiments. ** P < 0.01, *** P < 0.001 significantly different from the control group of Ba/F3‐NPM‐ALK transfected with scrambled siRNA. # P < 0.05, ## P < 0.01, ### P < 0.01 significantly different from the group of Ba/F3‐NPM‐ALK transfected with EBP2 siRNA #1 or EBP2 siRNA #2. (B) Total RNA was prepared and the expression of p21 mRNA was analyzed by quantitative real‐time PCR ( n = 3). Rpl13a mRNA was analyzed as an internal control. Error bars represent the SD of the mean. ** P < 0.01; *** P < 0.001 significantly different from the group of Ba/F3‐NPM‐ALK transfected with scrambled siRNA. ## P < 0.01 significantly different from the group of Ba/F3‐NPM‐ALK transfected with EBP2 siRNA #1 or EBP2 siRNA #2.

Article Snippet: Transduced Ba/F3 cells and Ki‐JK cells were transfected with siRNA by electroporation using the Neon transfection system (WAKENYAKU Co., Ltd., Kyoto, Japan) according to the manufacturer's instructions.

Techniques: Knockdown, Activation Assay, Expressing, Transfection, Control, Phospho-proteomics, Real-time Polymerase Chain Reaction

Journal: Molecular Oncology

Article Title: EBP2, a novel NPM‐ALK‐interacting protein in the nucleolus, contributes to the proliferation of ALCL cells by regulating tumor suppressor p53

doi: 10.1002/1878-0261.12822

Figure Lengend Snippet: Knockdown of EBP2 activates p53 through Akt in ALCL patient‐derived Ki‐JK cells. Ki‐JK cells were transfected with scrambled siRNA, EBP2 siRNA #1, or EBP2 siRNA #2. Twenty‐four hours after transfection, cells were treated with GDC‐0068 (2.5 and 5 μ m ) for 48 h. (A) Whole‐cell lysates were immunoblotted with an anti‐phospho‐Akt (Ser473), anti‐Akt, anti‐phospho‐p53 (Ser15), anti‐p53, anti‐p21, anti‐EBP2, or anti‐β‐actin antibody. The relative phosphorylation or expression levels of Akt, p53, p21 and EBP2 are shown in the graphs. Results represent the mean ± SD of three independent experiments. ** P < 0.01, *** P < 0.001 significantly different from the control group of Ki‐JK cells transfected with scrambled siRNA. # P < 0.05, ## P < 0.01, ### P < 0.001 significantly different from the control group of Ki‐JK cells transfected with EBP2 siRNA #1 or EBP2 siRNA #2. (B) Total RNA was prepared, and the expression of p21 mRNA was analyzed by quantitative real‐time PCR ( n = 3). Rpl13a mRNA was analyzed as an internal control. Error bars represent the SD of the mean. ** P < 0.01, *** P < 0.001 significantly different from the group of Ki‐JK cells transfected with scrambled siRNA. ## P < 0.01 significantly different from the group of Ba/F3‐NPM‐ALK transfected with EBP2 siRNA #1 or EBP2 siRNA #2.

Article Snippet: Transduced Ba/F3 cells and Ki‐JK cells were transfected with siRNA by electroporation using the Neon transfection system (WAKENYAKU Co., Ltd., Kyoto, Japan) according to the manufacturer's instructions.

Techniques: Knockdown, Derivative Assay, Transfection, Phospho-proteomics, Expressing, Control, Real-time Polymerase Chain Reaction

Journal: Molecular Oncology

Article Title: EBP2, a novel NPM‐ALK‐interacting protein in the nucleolus, contributes to the proliferation of ALCL cells by regulating tumor suppressor p53

doi: 10.1002/1878-0261.12822

Figure Lengend Snippet: Knockdown of EBP2 activates p53 through mTORC1 pathway in ALCL patient‐derived Ki‐JK cells. Ki‐JK cells were transfected with scrambled siRNA, EBP2 siRNA #1, or EBP2 siRNA #2. Twenty‐four hours after transfection, cells were treated with rapamycin (1 and 10 n m ) for 48 h. (A) Whole‐cell lysates were immunoblotted with an anti‐phospho‐p70 S6 kinase (Thr389), anti‐p70 S6 kinase, anti‐phospho‐p53 (Ser15), anti‐p53, anti‐p21, anti‐EBP2, or anti‐β‐actin antibody. The relative phosphorylation or expression levels of p70 S6 kinase, p53, p21, and EBP2 are shown in the graphs. Results represent the mean ± SD of three independent experiments. ** P < 0.01, *** P < 0.001 significantly different from the control group of Ki‐JK cells transfected with scrambled siRNA. # P < 0.05, ## P < 0.01, ### P < 0.001 significantly different from the control group of Ki‐JK cells transfected with EBP2 siRNA #1 or EBP2 siRNA #2. (B) Total RNA was prepared and the expression of p21 mRNA was analyzed by quantitative real‐time PCR ( n = 3). Rpl13a mRNA was analyzed as an internal control. Error bars represent the SD of the mean. ** P < 0.01 significantly different from the group of Ki‐JK cells transfected with scrambled siRNA. ## P < 0.01 significantly different from the group of Ba/F3‐NPM‐ALK transfected with EBP2 siRNA #1 or EBP2 siRNA #2.

Article Snippet: Transduced Ba/F3 cells and Ki‐JK cells were transfected with siRNA by electroporation using the Neon transfection system (WAKENYAKU Co., Ltd., Kyoto, Japan) according to the manufacturer's instructions.

Techniques: Knockdown, Derivative Assay, Transfection, Phospho-proteomics, Expressing, Control, Real-time Polymerase Chain Reaction

Journal: Progress in retinal and eye research

Article Title: CRISPR-CAS9 GENOME ENGINEERING: TREATING INHERITED RETINAL DEGENERATION

doi: 10.1016/j.preteyeres.2018.03.003

Figure Lengend Snippet: A-B: Immunohistochemical analysis of GFP expression (green) following transduction of human retinal explants with AAV5-GFP at 1 week post-subretinal delivery. Unlike in vitro HEK293 transfection efficiency, which is near 100%, AAV5 based gene delivery vectors typically transduce less that 50% of the photoreceptor cells targeted. C: In vitro NHEJ efficiency. Of 40 sgRNAs targeting ten independent genes associated with inherited retinal degenerative disease, an average NHEJ efficiency of 20.6 ± 1.3% NHEJ was detected.

Article Snippet: Also, specialized instrumentation, such as the NEON transfection system or Lonza’s Nucleofector, are required.

Techniques: Immunohistochemical staining, Expressing, Transduction, In Vitro, Transfection