Journal: Genes

Article Title: Heterozygous Knockout of ARID4B Using CRISPR/Cas9 Attenuates Some Aggressive Phenotypes in a Breast Cancer Cell Line.

doi: 10.3390/genes14122184

Figure Lengend Snippet: Figure 2. Construction and validation of the CRISPR/Cas9 system. (A) The structure of the ARID4B protein with a total of 1312 amino acids and the site where the mutations were generated in position 939, indicated as a hotspot. (B) Design of the sgRNA and validation of the ligation in the pX459 vector by Sanger sequencing. Only underlined region is shown. Red for T, Black for G, Blue for C, and Green for A. (C) Confirmation of the presence of genetic editions in the pool of MCF-7 cells after transfection. The surveyor assay produces a cut in the PCR product, generating two fragments indicated by arrows. (D) Validation of the presence of genetic editions in isolated clones. Here, 3, 10, and 21 represent the clones positive for mutations in ARID4B.

Article Snippet: Both oligos were aligned and cloned in Bbs1 digested pX459 (62988, Addgene, Cambridge, MA, USA).

Techniques: Biomarker Discovery, CRISPR, Generated, Ligation, Plasmid Preparation, Sequencing, Transfection, Isolation, Clone Assay

Journal: eLife

Article Title: Integrated transcriptome and proteome analysis reveals posttranscriptional regulation of ribosomal genes in human brain organoids

doi: 10.7554/eLife.85135

Figure Lengend Snippet: ( A ) RNA-FISH for the 5’TOP transcripts RPL5 and RPL11 in 40-day-old organoids (H9 background). Images show a typical ventricular zone (VZ)-like structure in the brain organoid with a zoomed-in image of the boxed area on the right. Dotted line marks the apical side of VZ. Scale bar = 50 µm. ( B ) Immunostaining for the 5’TOP proteins RPL5 and RPL11 in 40-day-old organoids (H9 background). Images show a typical VZ-like structure in the brain organoid with a zoomed-in image of the boxed area on the right. Dotted line marks the apical side of VZ. Scale bar = 50 µm. ( C ) Schematic representation of the dox-inducible 5’TOP (blue) and non-5’TOP (orange) tagBFP reporter constructs. ( D,E ) Representative cumulative distribution of reporter tagBFP levels in neural progenitors ( D ) and in neurons at day 40 ( E ) from 5’TOP reporter (blue) and non-5’TOP reporter (orange). ( F ) Confocal scan of a typical VZ-like structure in a 40-day-old organoid (H9 background) expressing G3BP1-mScarlet stained for RPL5 and RPL11 transcripts by RNA-FISH. Zoomed-in image of the inlay shows G3BP1 granules pointed by yellow arrows. Dotted line marks the apical side of VZ. Scale bar = 20 µm. ( G ) Quantification of RPL5 and RPL11 RNA-FISH signal intensities in the nucleus, G3BP1-positive granules and cytosol of progenitors in the VZ. p-Value of Mann-Whitney test. Each dot represents a cell. (Number of cells analyzed: RPL5: n=45; RPL11: n=34.)

Article Snippet: For SOX2 and TSC2 guides, these ds-DNA fragments were cloned into pSpCas9(BB)-2A-GFP plasmid (Addgene, 48138, RRID: Addgene_48138 ), modified to express eCas9 instead of WT-Cas9 and dTomato instead of GFP , using the Bbs1 cloning strategy of . For G3BP1 guide, the ds-DNA fragments were cloned into pSpCas9(BB)-2A-Puro (PX459) (Addgene, 62988, RRID: Addgene_62988 ).

Techniques: Immunostaining, Construct, Expressing, Staining, MANN-WHITNEY

Journal: eLife

Article Title: Integrated transcriptome and proteome analysis reveals posttranscriptional regulation of ribosomal genes in human brain organoids

doi: 10.7554/eLife.85135

Figure Lengend Snippet: ( A ) Confocal scan of ventricular zone (VZ) in control and sodium arsenite-treated organoids (H9 background) stained with anti-G3BP1 antibody and DAPI. Zoomed-in image of the inlay shows G3BP1 granules marked by yellow arrows in VZ and neuronal zones. Scale bar = 50 µm. ( B ) Quantification of G3BP1 signal intensity in the granules observed in the control and sodium arsenite-treated organoids. p-Value statistic of t-test. Each dot represents a granule. (Number of organoids analyzed, control: n=3; NaAs: n=4; number of granules analyzed: control: n=31; NaAs: n=51.) ( C ) Quantification of average number of G3BP1 granules per cell in VZ and neuronal zone of 40-day-old control organoids. p-Value statistic of t-test. Each dot represents an area from an organoid. Quantification performed in three organoids with total field of views: n=8 and number of cells counted: NPCs: n=829; neurons: n=804. ( D ) Confocal scan of VZ in a control organoid (Rozh-5 background) stained with anti-G3BP1 antibody and DAPI. Zoomed-in image of the inlay shows G3BP1 granules marked by yellow arrows in VZ. Scale bar = 20 µm. ( E ) Confocal scan of VZ in a control and sodium arsenite-treated organoids (H9 background) stained with anti-phoshoEIF2a antibody. Scale bar = 100 µm. ( F ) Quantification of average fluorescent intensity of phoshoEIF2a in the VZs of control and sodium arsenite-treated organoids and control organoids stained only with secondary antibody (no primary) (H9 background). Error bars mark standard deviation. p-Value Mann-Whitney test is shown. Each dot represents a VZ (for no primary, n=28; for control, n=28; for NaAs treated, n=18). Data from two batches of organoid differentiation.

Article Snippet: For SOX2 and TSC2 guides, these ds-DNA fragments were cloned into pSpCas9(BB)-2A-GFP plasmid (Addgene, 48138, RRID: Addgene_48138 ), modified to express eCas9 instead of WT-Cas9 and dTomato instead of GFP , using the Bbs1 cloning strategy of . For G3BP1 guide, the ds-DNA fragments were cloned into pSpCas9(BB)-2A-Puro (PX459) (Addgene, 62988, RRID: Addgene_62988 ).

Techniques: Control, Staining, Standard Deviation, MANN-WHITNEY

Journal: eLife

Article Title: Integrated transcriptome and proteome analysis reveals posttranscriptional regulation of ribosomal genes in human brain organoids

doi: 10.7554/eLife.85135

Figure Lengend Snippet: ( A ) Results of genotyping PCR to check insertion of the G3BP1-mScarlet transgene in the G3BP1 locus. Samples include G3BP1 reporter human embryonic stem cells (hESCs), control H9 hEScs, and negative control. ( B ) Confocal scan of ventricular zone (VZ) in a 40-day-old G3BP1-mScarlet organoid stained with anti-G3BP1 antibody and DAPI. Zoomed-in images of the inlay show granules in VZ marked by mScarlet (gray) and G3BP1 (green). Scale bars = 20 µm ( i ), 5 µm (ii). ( C ) Quantification of percentage of total G3BP1 granules observed in a field that are located in the VZ region. Schematic on the left shows G3BP1 granules in the tissue and quantification strategy. ( D ) Quantification of RPL5 and RPL11 RNA-FISH signal intensities in the nucleus, G3BP1-positive granules and the cytosol of progenitors in the VZ of sodium arsenite-treated organoids. The p-values of Mann-Whitney tests are shown. Each dot represents a cell. (Number of granules analyzed: RPL5: n=51; RPL11: n=111.) Figure 4—figure supplement 5—source data 1. Genotyping PCR of G3BP1-mScarlet cell line.

Article Snippet: For SOX2 and TSC2 guides, these ds-DNA fragments were cloned into pSpCas9(BB)-2A-GFP plasmid (Addgene, 48138, RRID: Addgene_48138 ), modified to express eCas9 instead of WT-Cas9 and dTomato instead of GFP , using the Bbs1 cloning strategy of . For G3BP1 guide, the ds-DNA fragments were cloned into pSpCas9(BB)-2A-Puro (PX459) (Addgene, 62988, RRID: Addgene_62988 ).

Techniques: Control, Negative Control, Staining, MANN-WHITNEY

Journal: eLife

Article Title: Integrated transcriptome and proteome analysis reveals posttranscriptional regulation of ribosomal genes in human brain organoids

doi: 10.7554/eLife.85135

Figure Lengend Snippet:

Article Snippet: For SOX2 and TSC2 guides, these ds-DNA fragments were cloned into pSpCas9(BB)-2A-GFP plasmid (Addgene, 48138, RRID: Addgene_48138 ), modified to express eCas9 instead of WT-Cas9 and dTomato instead of GFP , using the Bbs1 cloning strategy of . For G3BP1 guide, the ds-DNA fragments were cloned into pSpCas9(BB)-2A-Puro (PX459) (Addgene, 62988, RRID: Addgene_62988 ).

Techniques: Control, Recombinant, DNA Extraction, Isolation, Flow Cytometry, Immunohistochemistry, RNA Extraction, Fractionation, Software, Fluorescence, Sequencing

Journal: The Journal of Cell Biology

Article Title: MyD88 oligomer size functions as a physical threshold to trigger IL1R Myddosome signaling

doi: 10.1083/jcb.202012071

Figure Lengend Snippet: CRISPR/Cas9 gene editing of the MyD88 gene locus with mEGFP and p38 signaling in EL4-MyD88-GFP cells. (A–D) Workflow and validation for CRISPR/Cas9 editing of the MyD88 gene locus with mEGFP. (A) Schematic of the MyD88 gene locus and HDR template designed to insert a mEGFP open reading frame immediately upstream of the stop codon. (B) FACS results of WT and CRISPR/Cas9-engineered MyD88-GFP EL4 cells. Overlaid box indicates a sorting gate to select EL4-MyD88-GFP cells. (C) PCR screening of CRISPR/Cas9 editing to select homozygous edited cell clones. Schematic shows the primer design for PCR amplification of genomic DNA to detect WT, heterozygous, and homozygous edited EL4 cells. Homozygous MyD88-GFP edited cell clones are labeled in red. Three homozygous clones were retained for Western blot analysis. See for uncropped blots. (D) Western blot analysis of three MyD88-GFP clones. Blots were probed with anti-MyD88, then stripped and reprobed with anti-GFP. (E) To assess the level of MAPK pathway activation, EL4 cells were fixed (60 min after SLB contact) and stained for MyD88-GFP (green) and phospho-p38 (magenta); DAPI staining of nuclei (blue). Cells were imaged with confocal microscopy. Schematic shows the position of the confocal micrograph slice. Only cells in contact with SLB functionalized with IL1β had increased phospho-p38 nuclear staining intensity. Scale bar, 5 mm. (F) Reconstructed axial view of cells shown in E showing the localization of MyD88 to the cell–SLB contact zone and phospho-p38 staining under IL1β stimulation. Scale bar, 1 mm. (G) Quantification of phospho-p38 staining intensity. Mean ± SD from n = 12 (with IL1β) and 20 cells (without IL1β). fluo, fluorescence; FSA-A, forward scatter area; MW, molecular weight.

Article Snippet: We designed complementary oligonucleotides to be ligated into Bbs1-digested S. pyogenes Cas9 and chimeric gRNA expression plasmid pX459v2.0 (pX459v2.0-pSpCas9[BB]-2A-Puro; Addgene; #62988) or pX459v2.0-HypaCas9 (pX459v2.0-HypaCas9-2A-Puro; Addgene; #108294). sgRNA oligonucleotides were ordered from Integrated DNA Technologies.

Techniques: CRISPR, Clone Assay, Amplification, Labeling, Western Blot, Activation Assay, Staining, Confocal Microscopy, Fluorescence, Molecular Weight

Journal: The Journal of Cell Biology

Article Title: MyD88 oligomer size functions as a physical threshold to trigger IL1R Myddosome signaling

doi: 10.1083/jcb.202012071

Figure Lengend Snippet: CRISPR/Cas9 gene editing IRAK4 or IRAK1 gene loci with mScarlet and analysis of MyD88-GFP and IRAK4/1-mScarlet colocalization. (A) Schematic of the IRAK4 (top) and IRAK1 (bottom) gene locus and HDR template designed to insert a mScarlet open reading frame immediately upstream of the stop codon. EL4 cells were electroporated with HDR and gRNA/Cas9 plasmids to simultaneously edit MyD88 and IRAK4 or IRAK1 gene loci. Dual edited cells were selected by FACS and PCR (see for workflow, and Materials and methods). (B) Western blot analysis of three MyD88-GFP/IRAK4-mScarlet–expressing EL4 clones. Lysates were probed with anti-IRAK4, anti-RFP, anti-MyD88, and anti-GFP to confirm editing and insertion of fluorescent protein open reading frames at both gene loci. All data presented in the manuscript were acquired with clone 3. (C) Western blot analysis of MyD88-GFP/IRAK1-mScarlet–expressing EL4 clone. Lysates were probed with anti-IRAK4, anti-RFP, anti-MyD88, and anti-GFP to confirm editing and insertion of mScarlet or mEGFP open reading frames at both gene loci. See and for uncropped blots. (D–G) Data shown from clonal MyD88-GFP/IRAK4-mScarlet–expressing EL4 cells (green, D and E) or IRAK1-mScarlet (orange, F and G). (D and F) Data points are the proportion of individual cells from independent experiments. Bars are experimental replicate means. (E and G) Vertical line in E and G is at 4.5× MyD88-GFP. (D) Percentage (%) of MyD88-GFP puncta that colocalizes with IRAK4-mScarlet, combined, and by MyD88-GFP lifetime (<50 s or ≥50 s) per cell across experimental replicates. Violin plot of the percent of MyD88-GFP puncta that is colocalized with IRAK4-mScarlet, combined, and categorized by lifetime (<50 s or ≥50 s). Few tracks recruit IRAK4 (“All”, n = cells, replicates 1–3: 2.4%, n = 30; 3.2%, n = 31; 3.3%, n = 30). It is especially evident in MyD88-GFP puncta that persist for <50 s (“<50 s”, n = cells, replicates 1–3: 1.0%, n = 30; 1.2%, n = 31; 1.1%, n = 30). However, MyD88-GFP puncta that persist for ≥50 s are more likely to recruit IRAK4 (“≥50 s”, n = cells, replicates 1–3: 28%, n = 30; 35%, n = 31; 32%, n = 30). (E) Maximum MyD88-GFP size of IRAK4-mScarlet colocalized and noncolocalized puncta across experimental replicates. Density plot of MyD88-GFP size categorized as colocalized (green) or noncolocalized (black) with IRAK4-GFP. MyD88-GFP puncta colocalized with IRAK4-mScarlet are brighter (mean colocalized versus not colocalized, n = puncta, replicates 1–3: 11× MyD88-GFP, n = 835 versus 3.1× MyD88-GFP, n = 29,072; 10× MyD88-GFP, n = 601 versus 3.3× MyD88-GFP, n = 17,221; 11.7× MyD88-GFP, n = 552 versus 3.3× MyD88-GFP, n = 17,854). (F) Percentage (%) of MyD88-GFP puncta that colocalize with IRAK1-mScarlet, combined and by MyD88-GFP lifetime (<50 s or ≥50 s) per cell across experimental replicates. Few tracks recruit IRAK1 (“All”, n = cells, replicates 1–3: 6.3%, n = 15; 1.5%, n = 32; 7.6%, n = 40). It is especially evident in MyD88-GFP puncta that persist for <50 s (“<50 s”, n = cells, replicates 1–3: 3.9%, n = 15; 0.74%, n = 32; 4.1%, n = 40). However, MyD88 puncta that persist for ≥50 s are more likely to recruit IRAK1 (“≥50 s”, n = cells, replicates 1–3: 41%, n = 15; 9.0%, n = 32; 59%, n = 40). (G) Maximum MyD88-GFP size of IRAK1-mScarlet colocalized and noncolocalized puncta across experimental replicates. Density plot of MyD88-GFP size categorized as colocalized (orange) or not colocalized (black) with IRAK1-GFP. MyD88-GFP puncta colocalized with IRAK1-mScarlet are brighter (mean colocalized versus not colocalized, n = puncta, replicates 1–3: 19× MyD88-GFP, n = 291 versus 5.6× MyD88-GFP, n = 5,401; 6.2× MyD88-GFP, n = 314 versus 2.4× MyD88-GFP, n = 14,125; 10× MyD88-GFP, n = 1,439 versus 3.1× MyD88-GFP, n = 16,834). -ve, negative; +ve, positive; MW, molecular weight.

Article Snippet: We designed complementary oligonucleotides to be ligated into Bbs1-digested S. pyogenes Cas9 and chimeric gRNA expression plasmid pX459v2.0 (pX459v2.0-pSpCas9[BB]-2A-Puro; Addgene; #62988) or pX459v2.0-HypaCas9 (pX459v2.0-HypaCas9-2A-Puro; Addgene; #108294). sgRNA oligonucleotides were ordered from Integrated DNA Technologies.

Techniques: CRISPR, Western Blot, Expressing, Clone Assay, Molecular Weight

Journal: The Journal of Cell Biology

Article Title: MyD88 oligomer size functions as a physical threshold to trigger IL1R Myddosome signaling

doi: 10.1083/jcb.202012071

Figure Lengend Snippet: CRISPR/Cas9 KO IRAK4 and IRAK1 EL4 cell lines, and MyD88-GFP dynamics in KO cell lines. (A and B) Validation of IRAK4 and IRAK1 KO cell lines. Western blot analysis of two monoclonal EL4 IRAK4 KO (A) and IRAK1 KO (B) cell lines. (C) The percentage per cell of large MyD88 oligomers in WT, IRAK4 KO, and IRAK1 KO EL4 cell lines. Quantification of the percentage (%) per cell of MyD88-GFP puncta with a maximum intensity ≥4.5× GFP. Violin plots show the distribution of the cell data. Data points superimposed on violin plots are the averages of independent experimental replicates. Bars represent mean ± SEM ( n = 3 or 4 experimental replicates, with >10 cells measured per replicate). (D) The percentage per cell of large MyD88 oligomers is equivalent for short- and long-lived MyD88-GFP puncta across WT and KO cell lines. Quantification of the proportion (%) per cell of MyD88-GFP puncta with a maximum intensity of ≥4.5× GFP categorized by lifetimes of <50 s or ≥50 s. Violin plots show the distribution of the cell data. Data points superimposed on the violin plots are the averages from independent experiments. Bars represent mean ± SEM ( n = 3 or 4 experimental replicates, with >10 cells measured per replicates). WT and KO image means were compared using an unpaired Student's t test. (E) IL2 release in EL4-WT cells treated with IRAK4 inhibitor. EL4 cells were pretreated with DMSO or IRAK4 inhibitor (20 µm) for 30 min. Cells were then left untreated or stimulated with 10 ng/ml of IL1 for a further 24 h. IL2 release was assayed by ELISA. Values for IRAK4 inhibitor cells shown relative to DMSO-only treated cells. Average values calculated from three independent experiments. Bars represent mean ± SEM. (F) Quantification of the maximum intensity of MyD88-GFP puncta per cell with lifetimes of <50 s or ≥50 s. Violin plots show the distribution of individual cell measurements. Colored dots superimposed on violin plots correspond to the average value in the independent experiments ( n = 3 experimental replicates; encompasses measurements from 19–39 cells). Bars represent mean ± SEM. P values were calculated using a two-tailed unpaired Student’s t test. (G) Distribution of lifetimes for tracked MyD88-GFP puncta with a maximum intensity of <4.5× or ≥4.5× GFP in DMSO control ( n = 91,876 for <4.5× GFP and n = 4,836 for ≥4.5× GFP), and IRAK4 inhibitor ( n = 64,822 for <4.5× GFP and n = 3,828 for ≥4.5× GFP) –treated cells. MyD88-GFP puncta lifetimes collated from three experimental replicates. Max, maximum; Norm., normalized; MW, molecular weight.

Article Snippet: We designed complementary oligonucleotides to be ligated into Bbs1-digested S. pyogenes Cas9 and chimeric gRNA expression plasmid pX459v2.0 (pX459v2.0-pSpCas9[BB]-2A-Puro; Addgene; #62988) or pX459v2.0-HypaCas9 (pX459v2.0-HypaCas9-2A-Puro; Addgene; #108294). sgRNA oligonucleotides were ordered from Integrated DNA Technologies.

Techniques: CRISPR, Western Blot, Enzyme-linked Immunosorbent Assay, Two Tailed Test, Molecular Weight