Journal: eLife

Article Title: Dual control of pcdh8l/PCNS expression and function in Xenopus laevis neural crest cells by adam13/33 via the transcription factors tfap2α and arid3a

doi: 10.7554/eLife.26898

Figure Lengend Snippet: ( A ) Schematic representation of full length pcdh8l. A Flag tag (DYKDDDDK) was introduced just before the first cadherin repeat (EC1). The yellow arrow indicates the predicted cleavage site of adam13 based on the molecular weight of N- and C- terminal fragments. Red and blue asterisk indicate N and O glycosylation site, respectively. The monoclonal antibody 2F4 was produced against the cytoplasmic domain (Red). ( B ) Western blot from transfected Hek293T cells. Glycoproteins from the conditioned media were purified on concanavalin-A agarose. The cleavage fragment was detected using the N-terminus Flag tag with the mAb M2. Total pcdh8l and adam13 were detected using mAb 2F4 and mAb 4A7, respectively. The Pro ( P ) and Metalloprotease ( M ) active forms of adam13 are indicated. ( C ) Histogram representing the percentage of embryos lacking fluorescent neural crest cell in the migration pathway. ( D ) Photographs of representative embryos with or without fluorescent migrating neural crest cells. Embryos were injected at the one-cell stage with a morpholino targeting adam13 (10 ng MO13). Messenger RNA for RFP alone or combined with the different constructs was injected at the 8-cell-stage in a dorsal animal blastomere. Each injection was compared to RFP injected in control embryos in which RFP positive cranial neural crest cells have successfully migrated into the branchial and hyoid arches (0% inhibition). N = number of embryos scored from three or more independent experiments. Error bars represent standard error to the mean. One-way ANOVA was performed to determine statistical significance. Statistically significant at *p<0.05, ***p<0.005. 10.7554/eLife.26898.005 Figure 1—source data 1. Source data for .

Article Snippet: The identity was confirmed by detecting the recombinant Xenopus Rpn1 transfected in Hek293T cells (ATCC, CRL-3216 RCB2202).

Techniques: FLAG-tag, Molecular Weight, Glycoproteomics, Produced, Western Blot, Transfection, Purification, Migration, Injection, Construct, Control, Inhibition

Journal: eLife

Article Title: Dual control of pcdh8l/PCNS expression and function in Xenopus laevis neural crest cells by adam13/33 via the transcription factors tfap2α and arid3a

doi: 10.7554/eLife.26898

Figure Lengend Snippet: ( A ) Western blot from total membrane extracted from 10 embryos at gastrula stage (Stage 10) and early tailbud stage (Stage 20) detected using mAb2F4. ( B–C ) Cranial neural crest explants on fibronectin. Explants were left to migrate for 5 hr and fixed in 1XMBS, 3.7% formaldehyde for 1 hr, permeabilized for 30 min in 1XMBS containing 0.5% TritonX100. Staining with mAb 2F4 ( B ) clearly shows a membrane staining while DAPI stains the nuclei ( C ). ( D ) Wholemount immunostaining of tailbud stage embryo fixed in DENTS (20% DMSO 80% Methanol) using mAb 2F4. The red arrows indicate the tip of the cranial neural crest segments. The staining is identical to the one obtained by in situ hybridization with the pcdh8l probe. ( E ) Western blot on Hek293T cells extract transfected with the various Xenopus laevis protocadherin. The mAb 2F4 only recognizes pcdh8l (PCNS).

Article Snippet: The identity was confirmed by detecting the recombinant Xenopus Rpn1 transfected in Hek293T cells (ATCC, CRL-3216 RCB2202).

Techniques: Western Blot, Membrane, Staining, Immunostaining, In Situ Hybridization, Transfection

Journal: eLife

Article Title: Dual control of pcdh8l/PCNS expression and function in Xenopus laevis neural crest cells by adam13/33 via the transcription factors tfap2α and arid3a

doi: 10.7554/eLife.26898

Figure Lengend Snippet: ( A, C, D ) Relative expression of AP2α and pcdh8l in naïve ectoderm (animal cap) by real-time PCR. One-cell stage embryos were injected with 1 ng of the various adam13 constructs and morpholinos and embryos were collected at stage 9 to dissect animal cap cells (AC). AC were grown in 0.5X MBS until sibling embryos reached stage 20 to 22. Messenger RNA was extracted from 10 AC for gene expression analysis. Expression was normalized using GAPDH and compared to the expression in non-injected AC (NI). ( A ) Real-time PCR data show that adam13 can induce AP2α by more than four fold. Both proteolytic activity and the presence of the cytoplasmic domain are essential for full activation. ( B ) Luciferase activity of AP2α promoter in Hek293T cells shows induction by adam13 and ADAM19 but not ADAM9. For each transfection the luciferase values were normalized to the Renilla values driven by the CMV promoter. In these assays, the absence of proteolytic activity (A13E/A) reduced AP2α induction only slightly, while the deletion of the cytoplasmic domain (ΔCyto) prevented the activity. ( C ) Induction of AP2α by adam13 was prevented by the KD of AP2α (10 ng of MOAP2α) but not β-catenin (20 ng of Moβ-catenin). ( D ) Expression of AP2α in response of adam13 also depends on AP2α but not β-catenin. Error bars represent standard error to the mean (Mean ±S.E.M). One-way ANOVA was performed to determine statistical significance. Statistically significant at **p<0.01, ***p<0.001. 10.7554/eLife.26898.015 Figure 5—source data 1. Source data for .

Article Snippet: The identity was confirmed by detecting the recombinant Xenopus Rpn1 transfected in Hek293T cells (ATCC, CRL-3216 RCB2202).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Injection, Construct, Gene Expression, Activity Assay, Activation Assay, Luciferase, Transfection

Journal: eLife

Article Title: Dual control of pcdh8l/PCNS expression and function in Xenopus laevis neural crest cells by adam13/33 via the transcription factors tfap2α and arid3a

doi: 10.7554/eLife.26898

Figure Lengend Snippet: ( A–B ) Relative expression of AP2α and pcdh8l in animal caps dissected from control embryos, or embryos injected with adam13 or adam13 and a morpholino to Smad2 (MOSmad2, 25 ng). ( C ) Luciferase assays in Hek293T cells show no induction of AP2α promoter activity by Smad2 (0.5 µg) or Smad 7 (0.5 µg). Smad2 does not increase adam13 activation of the AP2α promoter, while Smad7 does not reduce adam13 induction of AP2α promoter. Smad2 and Smad7 were transfected with or without adam13 (0.5 µg) along with AP2α promoter-luciferase and the pRL-CMV (100 ng, 10 pg). The ratio of luciferase to renilla was used to normalize each transfection to the empty vector control (CS2). Error bars represent standard error to the mean (Mean ± S.E.M). One-way ANOVA was performed to determine statistical significance. Statistically significant at **p<0.01, ***p<0.001. 10.7554/eLife.26898.022 Figure 8—source data 1. Source data for .

Article Snippet: The identity was confirmed by detecting the recombinant Xenopus Rpn1 transfected in Hek293T cells (ATCC, CRL-3216 RCB2202).

Techniques: Expressing, Control, Injection, Luciferase, Activity Assay, Activation Assay, Transfection, Plasmid Preparation

Journal: eLife

Article Title: Dual control of pcdh8l/PCNS expression and function in Xenopus laevis neural crest cells by adam13/33 via the transcription factors tfap2α and arid3a

doi: 10.7554/eLife.26898

Figure Lengend Snippet: Western blot from transfected Hek293T cells. ( A–B ) cytoplasmic ( C ) and nuclear ( N ) extracts from cells transfected with the empty vector (CS2), Arid3a-flag, adam13 (A13) or both. The blots were re-probed with the transcription factor YY1 as a nuclear marker and GAPDH as a cytoplasmic marker. The full-length Xenopus laevis Arid3a is detected at approximately 60 kDa (Arid3a Long). A shorter fragment is detected at about 40 kDa (Arid3a Short). ( A ) Co-transfection of adam13 with Arid3a increases the Arid3a protein level in the cytoplasm by 30% and the shorter fragment of Arid3a in the nucleus by five folds. ( B ) Co-transfection of Arid3a with the proteolytically inactive mutant adam13 (A13E/A) or the mutant lacking the cytoplasmic domain (A13ΔCyto) does not increase the shorter fragment in the nucleus. ( C ) Membrane extract from Hek293T cells transfected with Arid3a-flag and the adam13 constructs. Co-transfection of Arid3a with adam13 increases the intensity of the 40 kDa Arid3a fragment. This is not observed in the absence of the adam13 cytoplasmic domain. In contrast, a much more significant increase is observed when Arid3a is co-transfected with the A13E/A mutant.

Article Snippet: The identity was confirmed by detecting the recombinant Xenopus Rpn1 transfected in Hek293T cells (ATCC, CRL-3216 RCB2202).

Techniques: Western Blot, Transfection, Plasmid Preparation, Marker, Cotransfection, Mutagenesis, Membrane, Construct

Journal: iScience

Article Title: Genome editing is induced in a binary manner in single human cells

doi: 10.1016/j.isci.2022.105619

Figure Lengend Snippet: Measurement of the copy number of RBM20 in HEK293T cells (A) A hypothetical situation that emphasizes the importance of the analysis of genome editing outcomes at the single cell level. Cell populations 1 and 2 consist of cells with totally different genotypes individually. However, the total allelic frequencies of WT, HDR, and NHEJ are exactly the same for both populations. (B) Representative karyotypes of HEK293T cells with three and four chromosome 10s. (C) A CGH analysis of HEK293T cells in comparison to diploid human iPS cells. The relative CGH signal of HEK293T cells normalized to that of diploid iPS cells is shown throughout the genome. (D) CGH copy number peak assignment. In the CGH analysis, there were several peaks of the CGH signal ratio between HEK293T cells and diploid iPS cells based on the chromosomal numbers. The highest peak corresponded to three copies per cell, and was set as the baseline of the CGH log ratio between HEK293T cells and iPS cells. (E) Line of fit between the copy number and the CGH log ratio based on the peak assignment shown in (D). The copy number and the CGH log ratio showed a clear linear correlation. (F) Scattered plot of relative CGH signal of HEK293T cells in comparison to diploid human iPS cells around the RBM20 gene. The relative CGH signals of HEK293T cells normalized by that of iPS cells are represented by +. No microduplications or microdeletions were detected around the RBM20 locus.

Article Snippet: HEK293T cells (RCB2202), HeLa cells (RCB0007) and PC9 cells (RCB4455) are available at RIKEN BioResource Research Center ( https://cell.brc.riken.jp/en/ ) and WTC11 iPS cells (GM25256) are available at Coriell Institute for Medical Research ( https://www.coriell.org/ ).

Techniques: Comparison

Journal: iScience

Article Title: Genome editing is induced in a binary manner in single human cells

doi: 10.1016/j.isci.2022.105619

Figure Lengend Snippet: Schematic illustration of the experiments of this study (A) Design of genome editing in RBM20 as a model case in this study. We introduced the R636S (c.1906C>A) mutation using CRISPR-Cas9 and a single-stranded oligonucleotide donor DNA. The resulting HDR allele has a C to A single nucleotide substitution, whereas the NHEJ alleles have various insertions and deletions. (B) The experimental flow of this study. We expressed Cas9-T2A-EGFP together with the gRNA targeting RBM20 to label HEK293T cells in which Cas9 protein was expressed. Using a microfluidic cell sorter (On-chip Sort), we sorted EGFP+ cells. Then, the sorted cells were plated into four 96-well plates by the SPiS. Cells were cultured for 2 to 4 weeks and then the genome editing outcomes were analyzed by amplicon sequencing. (C) An imaging analysis of single cells dispensed by the SPiS. The SPiS aspirates cell suspension into a specialized microtip and takes two images with a 1-s interval. Only when the SPiS recognized a single cell, the content of the tip was dispensed into a well of a 96-well plate. Otherwise, the SPiS would take another aliquot of the cell suspension to repeat the process.

Article Snippet: HEK293T cells (RCB2202), HeLa cells (RCB0007) and PC9 cells (RCB4455) are available at RIKEN BioResource Research Center ( https://cell.brc.riken.jp/en/ ) and WTC11 iPS cells (GM25256) are available at Coriell Institute for Medical Research ( https://www.coriell.org/ ).

Techniques: Mutagenesis, CRISPR, Cell Culture, Amplification, Sequencing, Imaging, Suspension

Journal: iScience

Article Title: Genome editing is induced in a binary manner in single human cells

doi: 10.1016/j.isci.2022.105619

Figure Lengend Snippet: Genome editing outcomes in RBM20 in individual HEK293T cells edited by Cas9 (A) Genome editing outcomes in isolated clones derived from single HEK293T cells edited by Cas9. We repeated the experiment three times, and isolated more than 90 clones out of 384 cells plated in all three trials. Each bar represents one clone, and the proportions of WT (green), NHEJ (blue), HDR (red), and HDR + NHEJ (purple) in one clone are also shown in each bar. (B) Total allelic frequencies in genome edited HEK293T cells in the three experiments shown in (A). (C) Models of the distributions of HEK293T cell clones with different genome editing outcomes in the Cas9 No.3 experiment, if genome editing randomly occurred at the frequencies shown in (B) in HEK293T cells with three or four copies of RBM20. (D) Comparison of the proportions of WT and full NHEJ clones between the mathematical models with three and four copies of RBM20 and the actually observed cells. Values ±S.E. are shown (n = 3). Student’s t test was used to evaluate differences. ∗p <0.05 and ∗∗p <0.01.

Article Snippet: HEK293T cells (RCB2202), HeLa cells (RCB0007) and PC9 cells (RCB4455) are available at RIKEN BioResource Research Center ( https://cell.brc.riken.jp/en/ ) and WTC11 iPS cells (GM25256) are available at Coriell Institute for Medical Research ( https://www.coriell.org/ ).

Techniques: Isolation, Clone Assay, Derivative Assay, Comparison

Journal: iScience

Article Title: Genome editing is induced in a binary manner in single human cells

doi: 10.1016/j.isci.2022.105619

Figure Lengend Snippet: Genome editing outcomes in RBM20 in individual HEK293T cells edited by HypaCas9 (A) Genome editing outcomes in isolated clones derived from single HEK293T cells edited by HypaCas9 and the single-stranded donor DNA. We repeated the same experiment three times. Each bar represents one clone, and the proportions of WT (green), NHEJ (blue), HDR (red), and HDR + NHEJ (purple) in one clone are also shown in each bar. (B) Total frequencies of WT (green), NHEJ (blue), HDR (red), and HDR + NHEJ (purple) allelic frequencies in genome edited HEK293T cells in the three experiments shown in (A). (C) Comparison of total allelic frequencies of WT, NHEJ, HDR, and HDR + NHEJ between Cas9 and HypaCas9. Values ±S.E. are shown (n = 3). Student’s t test was used to evaluate differences. ∗p <0.05 and NS: not significantly different (p >0.1). (D) Models of the distributions of HEK293T cell clones with different genome editing outcomes in the HypaCas9 No.3 experiment, if genome editing randomly occurred at the frequencies shown in (B) in HEK293T cells with three or four copies of RBM20. (E) Comparison of the proportions of WT and full NHEJ clones between the mathematical models with three and four copies of RBM20 and the actually observed cells. Values ±S.E. are shown (n = 3). Student’s t test was used to evaluate differences. ∗∗p <0.01. (F) Comparison of the proportions of clones with partial editing by HDR and clones with HDR accompanied by NHEJ events between the models with three and four copies of RBM20 and the actually observed cells. Values ±S.E. are shown (n = 3). Student’s t test was used to evaluate differences. ∗p <0.05 and NS: not significantly different (p >0.1).

Article Snippet: HEK293T cells (RCB2202), HeLa cells (RCB0007) and PC9 cells (RCB4455) are available at RIKEN BioResource Research Center ( https://cell.brc.riken.jp/en/ ) and WTC11 iPS cells (GM25256) are available at Coriell Institute for Medical Research ( https://www.coriell.org/ ).

Techniques: Isolation, Clone Assay, Derivative Assay, Comparison

Journal: iScience

Article Title: Genome editing is induced in a binary manner in single human cells

doi: 10.1016/j.isci.2022.105619

Figure Lengend Snippet: Genome editing outcomes in ATP7B and GRN in individual HEK293T cells edited by HypaCas9 (A) Genome editing outcomes in isolated clones derived from single HEK293T cells edited by HypaCas9 and the single-stranded donor DNA targeting ATP7B. We repeated the same experiment three times. Each bar represents one clone, and the proportions of WT (green), NHEJ (blue), HDR (red), and HDR + NHEJ (purple) in one clone are also shown in each bar. (B) Comparison of the proportions of WT and full NHEJ clones between the mathematical models with three copies of ATP7B and the actually observed cells. Values ±S.E. are shown (n = 3). Student’s t test was used to evaluate differences. ∗p <0.05 and ∗∗p <0.01. (C) Comparison of the proportions of clones with partial editing by HDR and clones with HDR accompanied by NHEJ events between the models with three copies of ATP7B and the actually observed cells. Values ±S.E. are shown (n = 3). Student’s t test was used to evaluate differences. NS: not significantly different (p >0.1). (D) Genome editing outcomes in isolated clones derived from single HEK293T cells edited by HypaCas9 and the single-stranded donor DNA targeting GRN. We repeated the same experiment three times. Each bar represents one clone, and proportions of WT (green), NHEJ (blue), HDR (red), and HDR + NHEJ (purple) in one clone are also shown in each bar. (E) Comparison of the proportions of WT and full NHEJ clones between the mathematical models with three and four copies of GRN and the actually observed cells. Values ±S.E. are shown (n = 3). Student’s t test was used to evaluate differences. ∗p <0.05 and ∗∗p <0.01.

Article Snippet: HEK293T cells (RCB2202), HeLa cells (RCB0007) and PC9 cells (RCB4455) are available at RIKEN BioResource Research Center ( https://cell.brc.riken.jp/en/ ) and WTC11 iPS cells (GM25256) are available at Coriell Institute for Medical Research ( https://www.coriell.org/ ).

Techniques: Isolation, Clone Assay, Derivative Assay, Comparison

Journal: iScience

Article Title: Genome editing is induced in a binary manner in single human cells

doi: 10.1016/j.isci.2022.105619

Figure Lengend Snippet:

Article Snippet: HEK293T cells (RCB2202), HeLa cells (RCB0007) and PC9 cells (RCB4455) are available at RIKEN BioResource Research Center ( https://cell.brc.riken.jp/en/ ) and WTC11 iPS cells (GM25256) are available at Coriell Institute for Medical Research ( https://www.coriell.org/ ).

Techniques: Recombinant, Transfection, Library Quantification, Microarray, Control, Amplification, Sequencing, Cloning, Software, FACS, Digital PCR