Journal: Molecular Therapy. Methods & Clinical Development

Article Title: In Vitro and In Vivo Genetic Disease Modeling via NHEJ-Precise Deletions Using CRISPR-Cas9

doi: 10.1016/j.omtm.2020.10.007

Figure Lengend Snippet: Design and Use of CRISPR gRNAs in HEK293T Cell Line (A) Exon 9 of the human PKLR gene is represented. Vertical lines and arrows are reported mutations, corresponding to one or more than 1 nt affected, respectively. Horizontal dashed lines represent the two gRNAs used. Vertical dashed lines represent predicted cut sites of gRNAs selected. (B) Sorting of green fluorescence based on the ZsGreen present in the electroporated plasmids. A 530/40- versus 580/30-nm dot plot was used to distinguish ZsGreen fluorescence from autofluorescence. The figure represents the ZsGreen fluorescence of the cell electroporated with the plasmid DNA containing only the Cas9 and the ZsGreen cDNAs (C), or containing the Cas9, the ZsGreen cDNAs and the sequences of the guide I (I) or the guide II (II). (C) Surveyor assay of the DNA obtained from the cells electroporated with the plasmids containing no guide, guide I, guide II, or both guides together (I+II). The gene editing generates a characteristic band pattern for guide I (160 bp + 135 bp), for guide II (191 bp + 104 bp), and a mixed pattern when both are used at the same time (160 bp + 104 bp). (D) Scheme of the band sizes that appear in (C) showing the 31-bp deletion resulting after the simultaneous electroporation of guides I and II.

Article Snippet: The HEK293T cell line (human embryonic kidney cell line competent to replicate vectors carrying the SV40 T antigen; ATCC, CRL-3219) was cultured in Iscove’s modified Dulbecco’s medium (IMDM; Gibco), HyClone fetal bovine serum (10%; GE Healthcare), and penicillin/streptomycin (1%; Gibco).

Techniques: CRISPR, Fluorescence, Plasmid Preparation, Electroporation

Journal: Journal of Innovative Optical Health Sciences

Article Title: Real-time detection of LMP1/LMP1 interaction in MβCD-induced apoptosis of nasopharyngeal carcinoma cells using FRET method

doi: 10.1142/s1793545819500196

Figure Lengend Snippet: Fig. 1. Cytotoxicity of MCD in CNE1-LMP1 cells and CNE1 cells. (a) Viability of CNE1-LMP1 cells, (b) viability of CNE1 cells, and (c) di®erences of viability between CNE1-LMP1 cells and CNE1 cells were measured by MTT assays after 40 mM MCD treatment for 30 min at 37C. Data represents the mean SD of at least three experiments. Di®erences between two groups were compared by Student's t-test.

Article Snippet: Plasmids and transfection LMP1 vector (p1990 SV40-LMP1) was a gift from Bill Sugden (Addgene plasmid #26654), cyan °uorescent protein (CFP) vector (EX-NEG-M33) and 1950019-2 J. I nn ov .

Techniques:

Journal: Journal of Innovative Optical Health Sciences

Article Title: Real-time detection of LMP1/LMP1 interaction in MβCD-induced apoptosis of nasopharyngeal carcinoma cells using FRET method

doi: 10.1142/s1793545819500196

Figure Lengend Snippet: Fig. 2. Induction of apoptosis in NPC cells by MCD treatment. (a) Expression of LMP1 protein in CNE1 and CNE1-LMP1 cells were measured by western blotting. (b) CNE1 cells were treated with 40 mM MCD for 30 min. (c) CNE1-LMP1 cells were treated with 40 mM MCD for 30 min. (d) Di®erences of caspase-3 cleavage ratio between CNE1 and CNE1-LMP1 cells. (e) Di®erences of mean °uorescence intensity induced by caspase-3 cleavage between CNE1 and CNE1-LMP1 cells. Corresponding statistical results from at least 4000 cells. These images were representative of three independent experiments. Scale bar, 100 m.

Article Snippet: Plasmids and transfection LMP1 vector (p1990 SV40-LMP1) was a gift from Bill Sugden (Addgene plasmid #26654), cyan °uorescent protein (CFP) vector (EX-NEG-M33) and 1950019-2 J. I nn ov .

Techniques: Expressing, Western Blot

Journal: Journal of Innovative Optical Health Sciences

Article Title: Real-time detection of LMP1/LMP1 interaction in MβCD-induced apoptosis of nasopharyngeal carcinoma cells using FRET method

doi: 10.1142/s1793545819500196

Figure Lengend Snippet: Fig. 4. Fluorescence spectra of CNE1 cells expressing fusion proteins. (a) CNE1 cells co-transfected with CFP and YFP plasmids, (b) transfected with CFP-YFP plasmids (18AA) and (c) co-transfected with LMP1-CFP and LMP1-YFP plasmids. All spectra were normalized as percentage of CFP maximal emission peak. These spectra were representative of three independent experiments.

Article Snippet: Plasmids and transfection LMP1 vector (p1990 SV40-LMP1) was a gift from Bill Sugden (Addgene plasmid #26654), cyan °uorescent protein (CFP) vector (EX-NEG-M33) and 1950019-2 J. I nn ov .

Techniques: Fluorescence, Expressing, Transfection

Journal: Journal of Innovative Optical Health Sciences

Article Title: Real-time detection of LMP1/LMP1 interaction in MβCD-induced apoptosis of nasopharyngeal carcinoma cells using FRET method

doi: 10.1142/s1793545819500196

Figure Lengend Snippet: Fig. 3. NF-kB p65 nuclear translocation in CNE1 and CNE1- LMP1 cells treated with 40 mM MCD for 15 min. These images were representative of three independent experiments. Scale bar, 20 m.

Article Snippet: Plasmids and transfection LMP1 vector (p1990 SV40-LMP1) was a gift from Bill Sugden (Addgene plasmid #26654), cyan °uorescent protein (CFP) vector (EX-NEG-M33) and 1950019-2 J. I nn ov .

Techniques: Translocation Assay

Journal: Journal of Innovative Optical Health Sciences

Article Title: Real-time detection of LMP1/LMP1 interaction in MβCD-induced apoptosis of nasopharyngeal carcinoma cells using FRET method

doi: 10.1142/s1793545819500196

Figure Lengend Snippet: Fig. 5. Analysis of LMP1/LMP1 interaction by FRET method. FRET was measured in CNE1 cells co-expressing the LMP1-CFP and LMP1-YFP fusion proteins as described in materials and methods. (a) FRET images. The pseudo-color images depicted FRET e±ciency, calculated as FRET/CFP ratio, with the color indicating the relative value at each pixel. (b) FRET °uorescence spectra. Images and °uorescence spectra were representative of three independent experiments. Scale bar, 10 m.

Article Snippet: Plasmids and transfection LMP1 vector (p1990 SV40-LMP1) was a gift from Bill Sugden (Addgene plasmid #26654), cyan °uorescent protein (CFP) vector (EX-NEG-M33) and 1950019-2 J. I nn ov .

Techniques: Expressing

Journal: Journal of Innovative Optical Health Sciences

Article Title: Real-time detection of LMP1/LMP1 interaction in MβCD-induced apoptosis of nasopharyngeal carcinoma cells using FRET method

doi: 10.1142/s1793545819500196

Figure Lengend Snippet: Fig. 6. Schematic ¯gure showing that relationship among lipid rafts, LMP1/LMP1 interaction and cell apoptosis.

Article Snippet: Plasmids and transfection LMP1 vector (p1990 SV40-LMP1) was a gift from Bill Sugden (Addgene plasmid #26654), cyan °uorescent protein (CFP) vector (EX-NEG-M33) and 1950019-2 J. I nn ov .

Techniques: