Journal: iScience

Article Title: Dissection of the MEF2D-IRF8 transcriptional circuit dependency in acute myeloid leukemia

doi: 10.1016/j.isci.2022.105139

Figure Lengend Snippet: MEF2D is an AML-biased dependency (A) Violin plots of MEF2D CERES effects. (B) Competition-based proliferation assays in MOLM-13 (AML, upper left), MV4; 11 (AML, upper right), THP1 (AML, bottom left) and K562 (an acute (erythro)blastic transformation of prior CML, bottom right) cell lines stably expressing Cas9. Plotted are relative GFP/sgRNA + population normalized based on day 3 (n = 3–5, mean ± SEM). sgNeg, negative control. sgPCNA, positive control. (C) Scatterplot that shows a linear correlation between MEF2D’s CERE dependency scores and mRNA expression in leukemia (red) or other cancer cell lines (light gray) in the DepMap dataset. Each dot represents one cell line; the shaded regions indicate 95% confidence interval for the linear regression model. (D) Immunoblotting of MEF2D in indicated whole-cell lysates. MEF2D hi cell lines are labeled in purple. HCC, Hepatocellular carcinoma.

Article Snippet: For human Cas9 sgRNAs, the oligos were ligated with T4 DNA ligase (NEB B0202S) to either BsmBI digested LRG2.1 plasmid (Addgene: 108,098) or LRCherry2.1 (Addgene: 108,099) backbones co-expressing a GFP or Cherry fluorescent tag, respectively.

Techniques: Transformation Assay, Stable Transfection, Expressing, Negative Control, Positive Control, Western Blot, Labeling

Journal: iScience

Article Title: Dissection of the MEF2D-IRF8 transcriptional circuit dependency in acute myeloid leukemia

doi: 10.1016/j.isci.2022.105139

Figure Lengend Snippet: MEF2D and IRF8 are upregulated in AML carrying KMT2A -r through enhancer reactivation (A) Gene tracks of H3K27ac and IRF8 ChIP signals in MOLM-13, THP-1, HEL or K562 cells. (B) Gene tracks of H3K27ac CUT&RUN signals in primary AML cells, with the key oncogenic mutations labeled on the left. (C) Schematic of CRISPRi (KRAB-dCas9)-mediated epigenomic silencing at MEF2D locus. Red bars indicate sgRNA positions. (D) RT-qPCR analysis of mRNA expression of MEF2D in MOLM-13 cells stably expressing dCas9-KRAB and transduced with indicated sgRNAs in Figure 5 B harvested on 4 days post-infection. Relative mRNA levels were normalized to GAPDH levels. Plotted are the mean ± SEM (n = 3 for negative controls, n = 2 for MEF2D sgRNAs). (E) Volcano plots of RNA-seq data in MA4- (left) or MA9- (right) transformed human CD34 + HSPCs versus normal HSPCs isolated from BM, where KMT2A translocation was induced via CRISPR-Cas9 ( Secker et al., 2020 ). (F) Competition-based proliferation assays in MA9-FLT3 ITD (left) or MA9-NRAS G12D (right) cells, which were generated by retroviral transduction of KMT2A-AF9 followed by FLT3 FLT3 ITD and NRAS G12D into human CD34 + HSPC cells, respectively ( Wei et al., 2008 ; Wunderlich et al., 2013 ) (n = 3, mean ± SEM).

Article Snippet: For human Cas9 sgRNAs, the oligos were ligated with T4 DNA ligase (NEB B0202S) to either BsmBI digested LRG2.1 plasmid (Addgene: 108,098) or LRCherry2.1 (Addgene: 108,099) backbones co-expressing a GFP or Cherry fluorescent tag, respectively.

Techniques: Labeling, Quantitative RT-PCR, Expressing, Stable Transfection, Transduction, Infection, RNA Sequencing Assay, Transformation Assay, Isolation, Translocation Assay, CRISPR, Generated

Journal: iScience

Article Title: Dissection of the MEF2D-IRF8 transcriptional circuit dependency in acute myeloid leukemia

doi: 10.1016/j.isci.2022.105139

Figure Lengend Snippet:

Article Snippet: For human Cas9 sgRNAs, the oligos were ligated with T4 DNA ligase (NEB B0202S) to either BsmBI digested LRG2.1 plasmid (Addgene: 108,098) or LRCherry2.1 (Addgene: 108,099) backbones co-expressing a GFP or Cherry fluorescent tag, respectively.

Techniques: Recombinant, Magnetic Beads, Protease Inhibitor, Cell Viability Assay, Clone Assay, Purification, Isolation, Sequencing, Software

Journal: bioRxiv

Article Title: TAOK1 regulates chemo- and radiosensitivity in BRCA1/2-deficient tumors

doi: 10.1101/2025.10.14.682031

Figure Lengend Snippet: ( A ) Schematic outline of the kinome shRNA and whole genome CRISPR-Cas9 screens. ( B ) Volcano plot representing gene summary of MAGeCK analysis of the kinome shRNA screens in KB2P-1.21 cells treated with olaparib. ( C ) Volcano plot representing gene summary of MAGeCK analysis of the whole genome CRISPR-Cas9 screens in RPE1-hTERT BRCA1 -/- ;p53 -/- cells treated with 2 Gy IR. ( D, E ) Growth assays in KB2P-3.4 (D) or KB1P-G3 (E) Taok1 -/- cell lines treated with the indicated doses of olaparib (D) or IR (E). Statistical analysis was performed using two-way ANOVA followed by Dunnett’s test. ( F ) Representative images of growth assays in KB1P-G3 NT, Taok1 -/- with empty rescue construct (pOZ-empty) or Taok1 full cDNA rescue construct (pOZ-Taok1) treated with the indicated doses of olaparib. ( G ) Kaplan-Meier survival curve of mice transplanted with KB1P-4S organoids with NT or Taok1 sgRNAs and treated with olaparib. Statistical analysis was performed with the log-rank test. ( H, I ) Growth assays in KB1P-G3 Taok1 -/- cell lines complemented with indicated rescue construct. Cells were treated with indicated doses of olaparib (H) or IR (I). Statistical analysis was performed using two-way ANOVA followed by Dunnett’s test.

Article Snippet: For CRISPR-Cas9 editing of human MDA-MB-436 cell line, NT sgRNA and TAOK1 sgRNAs were cloned into pX330 vector (Addgene, #42230) with puromycin resistance cassette.

Techniques: shRNA, CRISPR, Construct

Journal: bioRxiv

Article Title: TAOK1 regulates chemo- and radiosensitivity in BRCA1/2-deficient tumors

doi: 10.1101/2025.10.14.682031

Figure Lengend Snippet: (A) Volcano plot representing gene summary of MAGeCK analysis of the whole genome CRISPR-Cas9 screens in RPE1-hTERT BRCA1 -/- ;p53 -/- cells treated with 1 Gy IR. ( B-E ) Western blot analysis of TAOK1 protein expression of NT and TAOK1 KO in KB2P-3.4 (B), KB1P-G3 including HA-tagged, Taok1 cDNA rescue (pOZ-Taok1) (C), MDA-MB-436 (D) and KB2P-1.21 (E) cell lines. ( F ) TIDE analysis of polyclonal KB2P-1.21 cells. ( G, H ) Growth assays in KB1P-G3 NT, Taok1 -/- and Taok1 rescue cells treated with the indicated doses of olaparib (G) or talazoparib (H). Statistical analysis was performed using two-way ANOVA followed by Dunnett’s test. ( I, J ) Growth assays in MDA-MB-436 NT and TAOK1 -/- cell lines treated with indicated doses of olaparib (I) or IR (J). Statistical analysis was performed using two-way ANOVA followed by Dunnett’s test. ( K, L ) Growth assays in KB2P-1.21 NT and Taok1 sgRNA cells treated with the indicated doses of olaparib (K) or IR (L). Statistical analysis was performed using two-way ANOVA followed by Dunnett’s test. ( M ) TIDE analysis of polyclonal KB1P-4S organoids. ( N ) Immunohistochemistry staining for TAOK1 of untreated BRCA1;p53-deficient mouse mammary tumors with NT or Taok1 sgRNA. ( O ) Olaparib response of KB1P-4S organoids transduced with NT or Taok1 sgRNAs. Statistical analysis was performed using two-way ANOVA followed by Dunnett’s test.

Article Snippet: For CRISPR-Cas9 editing of human MDA-MB-436 cell line, NT sgRNA and TAOK1 sgRNAs were cloned into pX330 vector (Addgene, #42230) with puromycin resistance cassette.

Techniques: CRISPR, Western Blot, Expressing, Immunohistochemistry, Staining, Transduction

Journal: bioRxiv

Article Title: TAOK1 regulates chemo- and radiosensitivity in BRCA1/2-deficient tumors

doi: 10.1101/2025.10.14.682031

Figure Lengend Snippet: ( A-C ) Quantification (A, B) and representative images (C) of growth assays in KB1P-G3B1 WT (A) and KB1P-G3 WT and Taok1 -/- cells treated with the indicated doses of CP 43 and olaparib. Statistical analysis was performed using two-way ANOVA followed by Dunnett’s test. ( D ) Western blot analysis of expression level of HA-tagged Taok1 rescue constructs. ( E ) In vitro kinase assay of indicated KB1P-G3 cell lines. Protein was isolated from KB1P-G3 cell lines by co-IP with HA-tag. NT cell line was used as negative control with no HA-tag while the other cell lines expressed an HA-tag with the indicated construct. Bars represent mean ± SD, statistical analysis was done with two-way ANOVA followed by Dunnett’s test. ( F, G ) Representative images of growth assay of KB1P-G3 NT, Taok1 -/- and Taok1 full cDNA rescue (pOZ-Taok1) or two different kinase mutants (pOZ-K57A, pOZ-D169A) and a double mutant (pOZ-K57A+D169A) treated with indicated doses of olaparib (F) or IR (G).

Article Snippet: For CRISPR-Cas9 editing of human MDA-MB-436 cell line, NT sgRNA and TAOK1 sgRNAs were cloned into pX330 vector (Addgene, #42230) with puromycin resistance cassette.

Techniques: Western Blot, Expressing, Construct, In Vitro, Kinase Assay, Isolation, Co-Immunoprecipitation Assay, Negative Control, Growth Assay, Mutagenesis

Journal: bioRxiv

Article Title: TAOK1 regulates chemo- and radiosensitivity in BRCA1/2-deficient tumors

doi: 10.1101/2025.10.14.682031

Figure Lengend Snippet: ( A ) Graph representing percentage of cells with micronuclei upon 0.5 µM olaparib treatment for 48 h. Bars are plotted as mean ± SD, statistical analysis was performed using ANOVA followed by Tukey’s multiple comparison test. ( B ) Quantification of RAD51 foci formation upon 10 µM olaparib. Bars represent mean ± SD, statistical analysis was performed using ANOVA followed by Tukey’s multiple comparison test. ( C ) Representative images of RAD51 foci formation upon 10 µM olaparib in Brca1 reconstituted KB1P-G3B1, KB1P-G3 and KB2P-3.4 cell lines with the indicated modifications. ( D ) Quantification of DSB spectrum assay in HEK293T cells treated with the indicated siRNAs. Each dot represents an experiment ran in duplicates, bars represent mean ± SD, statistical analysis was performed using ANOVA followed by Tukey’s multiple comparison test. ( E ) Growth assays of KB1P-G3 NT, Taok1 -/- and TAOK1 rescue cells treated with the indicated doses of cisplatin. Statistical analysis was performed using two-way ANOVA followed by Dunnett’s test.

Article Snippet: For CRISPR-Cas9 editing of human MDA-MB-436 cell line, NT sgRNA and TAOK1 sgRNAs were cloned into pX330 vector (Addgene, #42230) with puromycin resistance cassette.

Techniques: Comparison

Journal: bioRxiv

Article Title: TAOK1 regulates chemo- and radiosensitivity in BRCA1/2-deficient tumors

doi: 10.1101/2025.10.14.682031

Figure Lengend Snippet: ( A ) Quantification of RAD51 foci formation 3 h post 10 Gy IR. Bars represent mean ± SD, statistical analysis was performed using ANOVA followed by Tukey’s multiple comparison test. ( B ) Verification of siRNA KD efficiency in HEK293T DSB-Spectrum_V1 cell line. ( C ) Growth assays with MDA-MB-436 NT and TAOK1 KO cell lines treated with the indicated doses of cisplatin. Statistical analysis was performed using two-way ANOVA followed by Dunnett’s test. ( D, E ) Growth assays in KB1P-G3 NT, TAOK1 KO and Taok1 rescue cell lines treated with indicated doses of carboplatin (D) or oxaliplatin (E). Statistical analysis was performed using two-way ANOVA followed by Dunnett’s test.

Article Snippet: For CRISPR-Cas9 editing of human MDA-MB-436 cell line, NT sgRNA and TAOK1 sgRNAs were cloned into pX330 vector (Addgene, #42230) with puromycin resistance cassette.

Techniques: Comparison

Journal: bioRxiv

Article Title: TAOK1 regulates chemo- and radiosensitivity in BRCA1/2-deficient tumors

doi: 10.1101/2025.10.14.682031

Figure Lengend Snippet: (A) Representative images of IHC staining for TAOK1 on human tumor samples of prostate and lung showing variable nuclear and diffuse cytoplasmatic localization. ( B ) Representative images of IHC staining for TAOK1 on human tumor samples of prostate, bladder and ovary showing varying TAOK1 expression levels. ( C )Western blot analysis of KB1P-G3 NT samples of cytoplasmatic (C) and nuclear (N) fraction and Taok1 -/- whole cell lysate. ( D ) Representative images of immunofluorescence for cellular expression pattern of KB1P-G3 Taok1 -/- cells with HA-tagged pOZ-Taok1 rescue construct compared to pOZ-empty vector control. ( E ) Growth assay in KB1P-G3 NT, Taok1 -/- and Taok1 rescue cell lines treated with indicated doses of camptothecin. Statistical analysis was performed using two-way ANOVA followed by Dunnett’s test. ( F ) Growth assay in KB2P-3.4 NT and Taok1 -/- clones treated with indicated doses of JH-RE-06 with or without olaparib (0.1 µM). Statistical analysis was performed using two-way ANOVA followed by Tukey’s test. ( G ) DNA fiber analysis in indicated cell lines with or without 4 mM HU treatment (3 h). Bar represents mean of IdU/CldU ratio of at least 250 fibers, statistical analysis was done with ANOVA followed by Tukey’s multiple comparison test. (H) DNA fiber analysis in indicated cell lines treated with 10 µM olaparib 2h prior and while labelling with CldU and IdU, ssDNA was digested with S1 nuclease if indicated. Bar represents the mean of IdU track length of at least 250 fibers, statistical analysis was done with ANOVA followed by Tukey’s multiple comparison test.

Article Snippet: For CRISPR-Cas9 editing of human MDA-MB-436 cell line, NT sgRNA and TAOK1 sgRNAs were cloned into pX330 vector (Addgene, #42230) with puromycin resistance cassette.

Techniques: Immunohistochemistry, Expressing, Western Blot, Immunofluorescence, Construct, Plasmid Preparation, Control, Growth Assay, Clone Assay, Comparison

Journal: bioRxiv

Article Title: TAOK1 regulates chemo- and radiosensitivity in BRCA1/2-deficient tumors

doi: 10.1101/2025.10.14.682031

Figure Lengend Snippet: ( A ) Quantification of SIRF foci formation of HA-tagged TAOK1 with and without 2 mM HU. Bars represent mean ± SD, statistical analysis was performed using ANOVA followed by Tukey’s multiple comparison test. ( B ) Representative images of analysis shown in ( A ). ( C ) Volcano plot of mass spectrum analysis of TAOK1 co-IP samples from KB1P-G3 NT nuclear fraction compared to TAOK1 KO. Positive log2 fold change values indicate enrichment in the NT nuclear fraction compared to TAOK1 KO of four independent replicas. ( D ) Western blot of input and co-IP of KB1P-G3 NT and TAOK1 KO. Pulldown was performed with PCNA antibody. ( E ) Growth assay of KB1P-G3 NT, TAOK1 KO and TAOK1 rescue cells treated with topotecan at indicated doses. Statistical analysis was performed using two-way ANOVA followed by Dunnett’s test. ( F ) Growth assay of KB1P-G3 NT, TAOK1 KO and TAOK1 rescue cells treated with JH-RE-06 alone or in combination with olaparib. Statistical analysis was performed using two-way ANOVA followed by Tukey’s test.

Article Snippet: For CRISPR-Cas9 editing of human MDA-MB-436 cell line, NT sgRNA and TAOK1 sgRNAs were cloned into pX330 vector (Addgene, #42230) with puromycin resistance cassette.

Techniques: Comparison, Co-Immunoprecipitation Assay, Western Blot, Growth Assay

Journal: bioRxiv

Article Title: TAOK1 regulates chemo- and radiosensitivity in BRCA1/2-deficient tumors

doi: 10.1101/2025.10.14.682031

Figure Lengend Snippet: ( A ) Graph showing replication speed by total track length of untreated DNA fiber assay in KB1P-G3 NT and TAOK1 KO cells. Bar represents the mean of at least 250 fibers, statistical analysis was done with unpaired t-test. ( B ) DNA fiber analysis in indicated cell lines with or without 4 mM HU treatment. Bar represents the mean of IdU/CldU ratio of at least 300 fibers, statistical analysis was done with unpaired t-test. ( C ) DNA fiber analysis in indicated cell lines treated with 10 µM olaparib 2h prior and while labelling with CldU and IdU, ssDNA was digested with S1 nuclease if indicated. Bar represents the mean of IdU track length of at least 250 fibers, statistical analysis was done with ANOVA followed by Tukey’s multiple comparison test. ( D, E ) Quantification (D) and representative images (E) of immunofluorescence analysis of RPA foci formation in KB1P-G3 NT, TAOK1 KO and TAOK1 rescue cells upon olaparib treatment (10 µM for 16 h). Bars indicate the mean number of foci per nucleus of at least 1000 nuclei. Statistical analysis was done with ANOVA followed by Tukey’s multiple comparison test. (F) Western blot of input and co-IP of KB1P-G3 NT and TAOK1 rescue cells. Pulldown was performed with TRIM25 antibody. ( G, H ) Western blot of indicated KB1P-G3 (H) and MDA-MB-436 (I) cell lines for ISG15 protein levels. Cells were untreated or treated with 10 µM olaparib for 24 h. Vinculin was used as loading control. ( I, J ) Schematic predicting replication dynamics in presence (I) or absence of TAOK1 (J) in BRCA1/2-deficient cells. (I) TAOK1 interacts with PCNA and TRIM25 and does not allow alternative fork protection mechanisms or ssDNA gap repair in BRCA-deficient cells, leading to RF instability. (J) In the absence of TAOK1, increased ISG15 expression and potentially recruitment to RF activates fork protection and gap suppression mechanisms and thus, RF stability.

Article Snippet: For CRISPR-Cas9 editing of human MDA-MB-436 cell line, NT sgRNA and TAOK1 sgRNAs were cloned into pX330 vector (Addgene, #42230) with puromycin resistance cassette.

Techniques: Comparison, Immunofluorescence, Western Blot, Co-Immunoprecipitation Assay, Control, Expressing

Journal: bioRxiv

Article Title: TAOK1 regulates chemo- and radiosensitivity in BRCA1/2-deficient tumors

doi: 10.1101/2025.10.14.682031

Figure Lengend Snippet: ( A, B ) Representative images (A) and quantification (B) of immunofluorescence analysis of RPA foci formation in KB2P-3.4 NT and Taok1 -/- cells upon olaparib treatment (10 µM for 16 h). Bars indicate mean number of foci per nucleus of at least 1000 nuclei. Statistical analysis was done with ANOVA followed by Tukey’s multiple comparison test. ( C, D ) Graph representing ssDNA intensity of native BrdU incorporation in KB1P-G3 (C) and MDA-MB-436 (D) cell lines. Cells were treated with 0.75 µM olaparib for 5 h. Ordinary one-way ANOVA with Dunnett’s multiple comparison test was used for statistical analysis. ( E ) Representative images of PLA assay between TRIM25 and HA-tag in KB1P-G3 NT and Taok1 rescue cells. ( F ) Kaplan-Meier curve of survival probability of SCAN-B dataset treated with non-chemotherapy. Statistical analysis was done with log-rank test.

Article Snippet: For CRISPR-Cas9 editing of human MDA-MB-436 cell line, NT sgRNA and TAOK1 sgRNAs were cloned into pX330 vector (Addgene, #42230) with puromycin resistance cassette.

Techniques: Immunofluorescence, Comparison, BrdU Incorporation Assay

Journal: bioRxiv

Article Title: TAOK1 regulates chemo- and radiosensitivity in BRCA1/2-deficient tumors

doi: 10.1101/2025.10.14.682031

Figure Lengend Snippet: ( A, B ) Analysis of the SCAN-B dataset for TAOK1 high or low expression. Kaplan-Meier curves for survival probability of all patients (A) and patients treated with chemotherapy specifically (B). Risk tables are shown below. Statistical analysis was done with log-rank test.

Article Snippet: For CRISPR-Cas9 editing of human MDA-MB-436 cell line, NT sgRNA and TAOK1 sgRNAs were cloned into pX330 vector (Addgene, #42230) with puromycin resistance cassette.

Techniques: Expressing

Journal: The Journal of Cell Biology

Article Title: Transmission of integrin β7 transmembrane domain topology enables gut lymphoid tissue development

doi: 10.1083/jcb.201707055

Figure Lengend Snippet: Talin binding to the β7 cytoplasmic domain activates integrin α4β7. (A) Expression of talin and β7 in parental or talin KO β7-expressing Jurkat T cells. Top: Total expression by Western blot. Bottom: Surface expression by flow cytometry. The filled histograms represent untransfected Jurkat cells, whereas open histograms represent β7-expressing Jurkat cells or talin KO cells. (B) Binding of soluble MAdCAM-1 to β7 expressing Jurkat T cells or talin KO cells. PMA (100 nM) markedly increased binding to parental but not talin KO cells. Mn 2+ (0.5 mM) stimulated binding to both cell types. Stimulated cells were compared with resting (None) for each cell line using one-way ANOVA. (C) Adhesion of β7-expressing Jurkat T cells or Jurkat-talin KO cells to MAdCAM-1 substrate in the presence or absence of PMA (100 nM) under a wall shear stress of 2 dyn/cm 2 . Nontransfected Jurkat T cells (Jurkat) provided a negative control. Jurkat-β7-Talin KO or Jurkat were compared with the Jurkat-β7 for each condition using one-way ANOVA. (D) Structural model of the talin F3 domain in complex with integrin β7 tail (Arg 728 to Thr 766 ). Talin F3 domain is shown by a surface representation and colored by charge. A ribbon diagram of the docked β7 tail is highlighted in red. β7-Leu 758 and -Tyr 759 in the NPLY motif are shown as light blue–colored stick figures. (E) Soluble MAdCAM-1 binding to 293T cells transfected with WT or mutant α4β7 with or without THD cotransfection. Mn 2+ (0.5 mM) was used as a positive control for integrin activation. Nontransfected 293T cells (MOCK) provided a negative control. Mutant integrins were compared with the WT for each condition using one-way ANOVA. (F) Adhesion of 293T cells transfected with WT or mutant α4β7 with or without THD cotransfection on MAdCAM-1 under a wall shear stress of 2 dyn/cm 2 . Nontransfected 293T cells (MOCK) provided a negative control. Mutant integrins were compared with the WT for each condition using one-way ANOVA. (G) Binding of soluble MAdCAM-1 to 293T-α4β7 cells transfected with EGFP vector, EGFP-THD, EGFP-THD(L325R), or EGFP-THD(W359A). THD-stimulated cells were compared with vector control (α4β7 + EGFP vector) for each cell line using two-way ANOVA. MFI, mean fluorescent intensity. (H) Adhesion of 293T-α4β7 cells transfected with EGFP vector, EGFP-THD, EGFP-THD(L325R), or EGFP-THD(W359A) on MAdCAM-1 under a wall shear stress of 2 dyn/cm 2 . 293T cells transfected with THD only and nontransfected 293T cells (MOCK) were used as negative controls. THD-stimulated cells were compared with vector control (α4β7 + EGFP vector) for each cell line using one-way ANOVA. Error bars show means ± SD. n = 5. NS, P > 0.05; *, 0.01 < P < 0.05; **, 0.001 < P < 0.01; ***, P < 0.001.

Article Snippet: Mouse β7 and human talin single guide RNAs (sgRNAs) were constructed in vector pSpCas9n(BB)-2A-Puro (48141; Addgene; ).

Techniques: Binding Assay, Expressing, Western Blot, Flow Cytometry, Shear, Negative Control, Transfection, Mutagenesis, Cotransfection, Positive Control, Activation Assay, Plasmid Preparation, Control

Journal: The Journal of Cell Biology

Article Title: Transmission of integrin β7 transmembrane domain topology enables gut lymphoid tissue development

doi: 10.1083/jcb.201707055

Figure Lengend Snippet: Blocking talin-induced change in β7 TMD topology abolished α4β7 activation. (A) Sequence alignment of partial TMDs of integrin β3 and β7 subunits. β3 Pro mutant site Ala 711 is highlighted in red. The sites in β7 that were mutated to Pro (Leu 721 and Leu 723 ) are highlighted in green and Gly 722 is highlighted in pink and are projected onto a homology model of the α4β7 TMD (α4 in blue and β7 in red). (B) Adhesion of 293T cells transfected with β7 WT or mutations (L721P, G722P, or L723P) plus α4 on MAdCAM-1 under a wall shear stress of 2 dyn/cm 2 . Nontransfected 293T cells (MOCK) were used as a negative control (Ctrl). Mutant integrins were compared with the WT using one-way ANOVA. (C) Binding of soluble MAdCAM-1 to 293T cells transfected with WT α4β7 or α4 in combination with mutants (L721P, G722P, and L723P) in the presence or absence of THD. Nontransfected 293T cells (MOCK) were used as a negative control. Mutant integrins were compared with the WT for each condition using one-way ANOVA. Error bars show means ± SD. n = 5 (A and B) or 4 (C). *, 0.01 < P < 0.05; **, 0.001 < P < 0.01; ***, P < 0.001. MFI, mean fluorescence intensity. (D) Coimmunoprecipitation of THD with α4β7 WT or mutants. Lysates of 293T cells were transfected as in C, in combination with THD-GFP, and α4β7 was isolated by immunoprecipitation (IP). Precipitated proteins were analyzed by Western blotting with the indicated antibodies and confirmed similar THD association with the mutant and WT integrins. Data are representative of at least three independent experiments. Note that a shorter exposure time was used for the THD input. Molecular masses are given in kilodaltons. (E) A model of how a Pro mutation can prevent transmission of altered topology of the β7 TMD by talin. The complex formed between the β7 cytoplasmic tail/TMD (red) and cytoplasmic talin F3 domain (surface representation; colored by charge) alters the topology of the inner portion of the transmembrane helix, which is transmitted to the outer moiety, where it can disrupt the outer membrane clasp , resulting in destabilization of the α-β TMD complex and integrin activation. β7(L721P) breaks the TMD helix into two helices connected by a flexible kink; the flexible kink prevents transmission of the talin-induced change in intracellular TMD topology to stabilize the α-β TMD interaction and block talin-induced activation of integrin α4β7.

Article Snippet: Mouse β7 and human talin single guide RNAs (sgRNAs) were constructed in vector pSpCas9n(BB)-2A-Puro (48141; Addgene; ).

Techniques: Blocking Assay, Activation Assay, Sequencing, Mutagenesis, Transfection, Shear, Negative Control, Binding Assay, Fluorescence, Isolation, Immunoprecipitation, Western Blot, Transmission Assay, Membrane

Journal: The Journal of Cell Biology

Article Title: Transmission of integrin β7 transmembrane domain topology enables gut lymphoid tissue development

doi: 10.1083/jcb.201707055

Figure Lengend Snippet: Inhibiting talin-induced change in β7 TMD topology impairs agonist-induced α4β7 activation. (A) Binding of soluble MAdCAM-1 to Jurkat T cells stably expressing WT or mutant β7(L721P or L723P) with or without CXCL12 or PMA stimulation. Mutant integrins were compared with the WT for each condition using one-way ANOVA. Error bars show means ± SD. n = 5. NS, P > 0.05; **, 0.001 < P < 0.01; ***, P < 0.001. MFI, mean fluorescence intensity. (B) β7 cell surface expression on Jurkat T cells stably expressing WT or mutant β7(L721P or L723P). Mock-transfected cells are depicted in the filled histograms. (C) α4β7 was immunoprecipitated (IP) from lysates of CXCL12-stimulated cells or unstimulated cells depicted in A, and bound proteins were analyzed by Western blotting with the indicated antibodies. Data are representative of at least three independent experiments. Molecular masses are given in kilodaltons.

Article Snippet: Mouse β7 and human talin single guide RNAs (sgRNAs) were constructed in vector pSpCas9n(BB)-2A-Puro (48141; Addgene; ).

Techniques: Activation Assay, Binding Assay, Stable Transfection, Expressing, Mutagenesis, Fluorescence, Transfection, Immunoprecipitation, Western Blot

Journal: The Journal of Cell Biology

Article Title: Transmission of integrin β7 transmembrane domain topology enables gut lymphoid tissue development

doi: 10.1083/jcb.201707055

Figure Lengend Snippet: Suppressing talin-induced change in β7 TMD topology perturbed mouse lymphocyte homing to the gut. (A) Cell surface expression of α4β7 in TK1-β7 KO cells stably expressing WT β7 or mutant β7(L721P). (B) Binding of soluble mouse MAdCAM-1 to the cells shown in A with or without CXCL12 stimulation. The TK1-β7 KO cell was used as a negative control. Stimulated cells were compared with resting (None) for each cell line using one-way ANOVA. MFI, mean fluorescence intensity. (C and D) In vivo competitive homing of TK1-β7 KO cells that stably express WT β7(β7 WT) or proline mutant β7(β7[L721P]) to different lymphoid tissues. TK1-β7 WT or TK1-β7(L721P) cells were labeled with eFluor 670. TK1 parental (TK1) cells were labeled with CFSE as an input control. Equal numbers (2 × 10 7 ) of eFluor 670–labeled cells and CFSE-labeled cells were mixed and then intravenously injected into C57BL/6J mice. Lymphoid organs were isolated 2 h after injection. The eFluor 670– or CFSE-labeled cells that homed into different lymphoid organs were enumerated by flow cytometry. The total numbers of homed cells to different lymphoid organs are shown in C. MLN (per mouse), PP (per mouse), PLN (per lymph node), and SP (per mouse). The ratio of TK1 parental cells to TK1-KO cells reconstituted with β7 WT or β7(L721P) cells recovered from different lymphoid organs is shown in D. Error bars show means ± SD. n = 5 (A and B) or 24 (C and D). NS, P > 0.05; **, 0.001 < P < 0.01; ***, P < 0.001.

Article Snippet: Mouse β7 and human talin single guide RNAs (sgRNAs) were constructed in vector pSpCas9n(BB)-2A-Puro (48141; Addgene; ).

Techniques: Expressing, Stable Transfection, Mutagenesis, Binding Assay, Negative Control, Fluorescence, In Vivo, Labeling, Control, Injection, Isolation, Flow Cytometry

Journal: The Journal of Cell Biology

Article Title: Transmission of integrin β7 transmembrane domain topology enables gut lymphoid tissue development

doi: 10.1083/jcb.201707055

Figure Lengend Snippet: Disruption of GALT development in Itgb7 L720P/L720P mice. (A) Schematic of the Cas9/sgRNA-targeting sites in Itgb7 . The sgRNA-targeting sequences are underlined in blue, and the protospacer-adjacent motif (PAM) sequence is labeled in blue in the WT sequence. The MaeI restriction site removed from the WT and ApaI site introduced in the mutant are labeled as are the Leu codon changed to Pro. (B) Sequencing analysis of WT and β7(L720P) knock-in mice. DNA sequencing confirmed a leucine to proline substitution at position 720 of the mouse β7 integrin gene (position 721 in human β7 integrin gene). The mutation is labeled in red. The silent mutations are labeled in green and with green asterisks. (C) The absolute number of CD3 + T cells and B220 + B cells isolated from Itgb7 WT/WT mice and Itgb7 WT/L720P , or Itgb7 L720P/L720P littermates are shown. Error bars show means ± SD. n = 8. NS, P > 0.05; ***, P < 0.001.

Article Snippet: Mouse β7 and human talin single guide RNAs (sgRNAs) were constructed in vector pSpCas9n(BB)-2A-Puro (48141; Addgene; ).

Techniques: Disruption, Sequencing, Labeling, Mutagenesis, Knock-In, DNA Sequencing, Isolation

Journal: The Journal of Cell Biology

Article Title: Transmission of integrin β7 transmembrane domain topology enables gut lymphoid tissue development

doi: 10.1083/jcb.201707055

Figure Lengend Snippet: Itgb7 L720P/L720P adult mice have reduced gut-tropic β7 high effector/activated T cells. (A) Equivalent cell surface expression of integrin αL, α4, β1, β2, and β7 and intracellular expression of kindlin 3 and talin in neonatal Itgb7 WT/WT mice and Itgb7 L720P/L720P littermates are shown. (B) Cell surface expression of β7 is reduced in 8-wk-old Itgb7 L720P/L720P compared with Itgb7 WT/WT littermates. (C) The ratio of cell surface expression of integrin β7 on effector/activated T cells (CD62L low CD44 high ) compared with naive T cells (CD62L high CD44 low ) in 8-wk-old Itgb7 WT/WT mice and Itgb7 L720P/L720P littermates. Mean fluorescence intensities are displayed on the representative histograms. Error bars show means ± SD. **, 0.001 < P < 0.01. Teff, effector T cells.

Article Snippet: Mouse β7 and human talin single guide RNAs (sgRNAs) were constructed in vector pSpCas9n(BB)-2A-Puro (48141; Addgene; ).

Techniques: Expressing, Fluorescence

Journal: Bio-protocol

Article Title: Construction of Viral Vectors for Cell Type-specific CRISPR Gene Editing in the Adult Mouse Brain

doi: 10.21769/BioProtoc.3334

Figure Lengend Snippet: Our method consists of four steps: (1) crossing Cre-dependent spCas9 knockin mice with a Cre-driver mouse line to express spCas9 in the target neural populations, (2) cloning of sgRNA into the spCas9 expressing plasmid, (3) construction of an AAV vector tandemly expressing dual sgRNA, and (4) AAV packaging and stereotaxic viral injection into the target brain area.

Article Snippet: Pipette tips 24-well plate (coated) (Thermo Fisher Scientific, catalog number: 142475) 1 ml plastic tube NEB10-beta Competent E. coli (New England Biolabs, catalog number: C3019) pX458 (Addgene, catalog number: 48138) pAAV EF1α DIO mCherry (Addgene, catalog number: 20299) BbsI-HF (New England Biolabs, catalog number: R3539) MluI-HF (New England Biolabs, catalog number: R3198) DpnI (New England Biolabs, catalog number: R0176) Alkaline Phosphatase, Calf Intestinal (CIP) (New England Biolabs, catalog number: M0290) Guide oligonucleotides (see Procedure A) Quick ligation kit (New England Biolabs, catalog number: M2200) Gibson Assembly Master Mix (New England Biolabs, catalog number: E2611) PrimeSTAR HS DNA polymerase (Takara, catalog number: R010) dNTP Mix 10 mM each (the mix of 10 mM of dTTP, dCTP, dGTP and dATP) (GenScript, catalog number: {"type":"entrez-nucleotide","attrs":{"text":"C01689","term_id":"1433919","term_text":"C01689"}} C01689 ) QIAquick Gel Extraction kit (QIAGEN, catalog number: 28704) QIAprep Spin Miniprep kit (QIAGEN, catalog number: 27104) Tris-EDTA, (1x Solution) (Fisher BioReagents, catalog number: BP2473) LB Broth EZMix Powder (Sigma, catalog number: L7658) LB Agar (Fisher BioReagents, catalog number: BP1425) Ampicillin (Gemini Bio-Products, catalog number: 400-130P) Primer to verify the cloning of sgRNA into pX458: 5’-gactatcatatgcttaccgt-3’ Primer to verify the tandem cloning of sgRNAs into pAAV: 5’-actgacgggcaccggagcca-3’ Primers for Gibson Assembly: Primer S1: 5’-taggggttcctgcggccgcacgcgtgagggcctatttc-3’ Primer AS1: 5’-ataggccctctctagaaaaaaagcaccgactc-3’ Primer S2: 5’-tttttctagagagggcctatttcccatg-3’ Primer AS2: 5’-atccatctttgcaaagcttacgcgtaaaaaagcaccgac-3’ Optional (see Procedure C) NIH3T3 cell (ATCC, catalog number: CRL-1658) AAV vector (pX458-sgRNA or pX458 empty as negative control ) Primers: You can design the primers to amplify the target exons by CHOPCHOP (see Procedure C) PrimeSTAR HS DNA polymerase FuGENE6 Transfection Reagent (Promega, catalog number: E2693) DMEM (1x) (Gibco, catalog number: 11995-065) Fetal Bovine Serum (Corning, catalog number: 35-015-CV) Penicillin/Streptomycin (Gibco, catalog number: 15070-063) PBS, pH 7.4 (Gibco, catalog number: 10010-023) TrypLE Select (1x) (Gibco, catalog number: 12563-011) QuickExtract DNA Extraction Solution (Epicentre, catalog number: QE0905T) T7 Endonuclease I (New England Biolabs, catalog number: M0302)

Techniques: Knock-In, Clone Assay, Expressing, Plasmid Preparation, Injection

Journal: Bio-protocol

Article Title: Construction of Viral Vectors for Cell Type-specific CRISPR Gene Editing in the Adult Mouse Brain

doi: 10.21769/BioProtoc.3334

Figure Lengend Snippet: A. Cloning of guide oligonucleotides. Oligonucleotides with 20-nt guide sequence and overhangs for cloning into BbsI site in pX458 were annealed and cloned into BbsI-digested pX458. B. Cloning of U6 promoter-driven sgRNAs. U6 promoter followed by sgRNA1 or sgRNA2 were PCR amplified, respectively, by using primers with overhangs for Gibson Assembly. C. Gibson Assembly of the dual sgRNA virus vector. U6-gRNA1 and U6-gRNA2 were cloned into MluI-digested pAAV EF1α DIO mCherry using Gibson Assembly.

Article Snippet: Pipette tips 24-well plate (coated) (Thermo Fisher Scientific, catalog number: 142475) 1 ml plastic tube NEB10-beta Competent E. coli (New England Biolabs, catalog number: C3019) pX458 (Addgene, catalog number: 48138) pAAV EF1α DIO mCherry (Addgene, catalog number: 20299) BbsI-HF (New England Biolabs, catalog number: R3539) MluI-HF (New England Biolabs, catalog number: R3198) DpnI (New England Biolabs, catalog number: R0176) Alkaline Phosphatase, Calf Intestinal (CIP) (New England Biolabs, catalog number: M0290) Guide oligonucleotides (see Procedure A) Quick ligation kit (New England Biolabs, catalog number: M2200) Gibson Assembly Master Mix (New England Biolabs, catalog number: E2611) PrimeSTAR HS DNA polymerase (Takara, catalog number: R010) dNTP Mix 10 mM each (the mix of 10 mM of dTTP, dCTP, dGTP and dATP) (GenScript, catalog number: {"type":"entrez-nucleotide","attrs":{"text":"C01689","term_id":"1433919","term_text":"C01689"}} C01689 ) QIAquick Gel Extraction kit (QIAGEN, catalog number: 28704) QIAprep Spin Miniprep kit (QIAGEN, catalog number: 27104) Tris-EDTA, (1x Solution) (Fisher BioReagents, catalog number: BP2473) LB Broth EZMix Powder (Sigma, catalog number: L7658) LB Agar (Fisher BioReagents, catalog number: BP1425) Ampicillin (Gemini Bio-Products, catalog number: 400-130P) Primer to verify the cloning of sgRNA into pX458: 5’-gactatcatatgcttaccgt-3’ Primer to verify the tandem cloning of sgRNAs into pAAV: 5’-actgacgggcaccggagcca-3’ Primers for Gibson Assembly: Primer S1: 5’-taggggttcctgcggccgcacgcgtgagggcctatttc-3’ Primer AS1: 5’-ataggccctctctagaaaaaaagcaccgactc-3’ Primer S2: 5’-tttttctagagagggcctatttcccatg-3’ Primer AS2: 5’-atccatctttgcaaagcttacgcgtaaaaaagcaccgac-3’ Optional (see Procedure C) NIH3T3 cell (ATCC, catalog number: CRL-1658) AAV vector (pX458-sgRNA or pX458 empty as negative control ) Primers: You can design the primers to amplify the target exons by CHOPCHOP (see Procedure C) PrimeSTAR HS DNA polymerase FuGENE6 Transfection Reagent (Promega, catalog number: E2693) DMEM (1x) (Gibco, catalog number: 11995-065) Fetal Bovine Serum (Corning, catalog number: 35-015-CV) Penicillin/Streptomycin (Gibco, catalog number: 15070-063) PBS, pH 7.4 (Gibco, catalog number: 10010-023) TrypLE Select (1x) (Gibco, catalog number: 12563-011) QuickExtract DNA Extraction Solution (Epicentre, catalog number: QE0905T) T7 Endonuclease I (New England Biolabs, catalog number: M0302)

Techniques: Clone Assay, Sequencing, Amplification, Plasmid Preparation

Journal: Cancer cell

Article Title: A transcription factor addiction in leukemia imposed by the MLL promoter sequence

doi: 10.1016/j.ccell.2018.10.015

Figure Lengend Snippet: (A) Heatmap depicts the log2 fold-change of sgRNA abundance (averaging each independent sgRNA targeting a gene) after 14 population doublings. The MLL fusion-biased hits were identified and ranked by subtracting the average of log2 fold-change of the four MLLfusion cell lines from average log2 fold-change of five MLLWT leukemia cell lines. (B) Competition-based proliferation assay of individual sgRNAs performed in the indicated Cas9-expressing cell lines. sgRNA expression is linked to a GFP reporter. Plotted is the GFP% cells (normalized to the day 3 measurement) at the indicated timepoints during culturing. A sgRNA targeting the kinase domain of CDK1 was included as a positive control. sgZFP64 and sgMLL data are the average of two independent sgRNAs. (n=3) (C) Western blotting of ZFP64 or HSC70 (loading control) in whole cell lysates prepared from the indicated Cas9+ leukemia cell lines transduced with the indicated sgRNAs. Lysates were prepared on day 5 post-infection. ‘e’ represents the exon targeted by the sgRNA. (D) RNA-seq scatterplot analysis comparing fold-change of mRNA levels following MLL and ZFP64 knockout in MOLM-13, NOMO-1, and OCI-AML3 cells. Log2 transformed fold-change were calculated based on the effects of two independent sgRNAs targeting ZFP64 or MLL compared to Neg1 negative control sgRNA. RPKM, Reads Per Kilobase of transcript per Million mapped reads. Known MLL target genes are highlighted. All bar graphs represent the mean ± SEM. See also Figure S1.

Article Snippet: Plasmid construction and sgRNA cloning All the Cas9-positive cancer cell lines in this study were derived by lentiviral transduction with a Cas9 expression vector (EFS-Cas9-P2A-Puro, Addgene: 108100).

Techniques: Proliferation Assay, Expressing, Positive Control, Western Blot, Control, Transduction, Infection, RNA Sequencing, Knock-Out, Transformation Assay, Negative Control

Journal: bioRxiv

Article Title: Epicardial HDAC3 promotes myocardial growth through a novel microRNA pathway

doi: 10.1101/2021.09.16.460538

Figure Lengend Snippet: (A) Representative immunofluorescence staining of HDAC3 and WT1 in Hdac3 eko ( Hdac3 f/f ; Wt1 CreERT2/+ ) hearts and control (CTL; Hdac3 f/+ ; Wt1 CreERT2/+ ) hearts. Tamoxifen was given to dams intraperitoneally (150 mg/kg body weight) at E8.5 (scale bars, 25 μm). (B) Representative H&E staining of Hdac3 eko and CTL hearts. Quantification is shown on the right (* P <0.05 by Student ’ s t -test; scale bars: 250 μm). (C) Representative immunofluorescence staining BrdU and p-H3 in in Hdac3 eko hearts and CTL hearts. Quantification is shown on the right (* P <0.05 by Student ’ s t -test; scale bars: 250 μm).

Article Snippet: To generate stable Hdac3 KO MECs, we cloned Hdac3 sgRNA into lentiCRISPR v2 vector (Addgene #52961; Watertown, MA). sgRNA primers (targeting on Hdac3 Exon2) were 5 ’ -CACCGCATAGCCTAGTCCTGCATTA-3 ’ (forward) and 5 ’ -AAACTAATGCAGGA CTAGGCTATGC-3 ’ (reverse).

Techniques: Immunofluorescence, Staining

Journal: bioRxiv

Article Title: Epicardial HDAC3 promotes myocardial growth through a novel microRNA pathway

doi: 10.1101/2021.09.16.460538

Figure Lengend Snippet: (A) Generation of Hdac3 knockout (KO) and empty vector control (EV) MECs by CRISPR/CAS9. Deletion of Hdac3 was verified by western blot. (B) Gene ontology (GO) pathway analyses and (C) volcano plot of RNA Sequencing in Hdac3 KO and EV MECs. (D) Quantification of Fgf9 and Igf2 in Hdac3 KO MECs by qRT-PCR. Gapdh was used as cDNA loading control (* P <0.05 by Student ’ s t -test). (E) Quantification of FGF9 and IGF2 in Hdac3 KO and EV MECs by western blot. GAPDH was used as protein loading control (* P <0.05 by Student ’ s t -test).

Article Snippet: To generate stable Hdac3 KO MECs, we cloned Hdac3 sgRNA into lentiCRISPR v2 vector (Addgene #52961; Watertown, MA). sgRNA primers (targeting on Hdac3 Exon2) were 5 ’ -CACCGCATAGCCTAGTCCTGCATTA-3 ’ (forward) and 5 ’ -AAACTAATGCAGGA CTAGGCTATGC-3 ’ (reverse).

Techniques: Knock-Out, Plasmid Preparation, CRISPR, Western Blot, RNA Sequencing Assay, Quantitative RT-PCR

Journal: bioRxiv

Article Title: Epicardial HDAC3 promotes myocardial growth through a novel microRNA pathway

doi: 10.1101/2021.09.16.460538

Figure Lengend Snippet: (A) Secretion of FGF9 and IGF2 from Hdac3 KO and EV MECs. Coomassie brilliant blue staining of total extracted proteins from supernatants served as protein loading controls. FGF9 and IGF2 in the MEC supernatants were detected by western blot. Arrows point to the target bands. Quantifications are shown on the right. (B&C) The effects of MEC supernatants and/or recombinant FGF9 or IGF2 on E13.5 Tnnt2 nGFP/+ CM proliferation. Representative immunofluorescence micrographs are shown. Percentage of p-H3+ CMs and total number of CMs were quantitate (* P <0.05 by ANOVA followed by Bonferroni post-hoc test; scale bars: 275 μm).

Article Snippet: To generate stable Hdac3 KO MECs, we cloned Hdac3 sgRNA into lentiCRISPR v2 vector (Addgene #52961; Watertown, MA). sgRNA primers (targeting on Hdac3 Exon2) were 5 ’ -CACCGCATAGCCTAGTCCTGCATTA-3 ’ (forward) and 5 ’ -AAACTAATGCAGGA CTAGGCTATGC-3 ’ (reverse).

Techniques: Staining, Western Blot, Recombinant, Immunofluorescence

Journal: bioRxiv

Article Title: Epicardial HDAC3 promotes myocardial growth through a novel microRNA pathway

doi: 10.1101/2021.09.16.460538

Figure Lengend Snippet: (A) Decease of Fgf9 and Igf2 mRNAs after RGFP966 (a selective Hdac3 inhibitor) treatment. MECs were treated with 10 uM RGFP966 or vehicle for 24 hours. mRNA levels were quantified by qRT-PCR. Gapdh was used as a cDNA loading control (* P <0.05 by Student ’ s t -test). (B) Decease of FGF9 and IGF2 protein levels after RGFP966 treatment. MECs were treated with 5 or 10 uM RGFP966 or vehicle for 24 hours. FGF9 and IGF2 were quantified by western blot. GAPDH was used as protein loading control (* P <0.05 by Student ’ s t -test). (C) mRNA levels of Fgf9 and Igf2 in Hdac3 KO and EV MECs after 24-hour treatment with Hdac3 WT, Y298H mutant, or mCherry (CTL) lentivirus. mRNAs were quantified by qRT-PCR ( n =3, * P <0.05 when compared to the CTL group by ANOVA followed by Bonferroni post-hoc test).

Article Snippet: To generate stable Hdac3 KO MECs, we cloned Hdac3 sgRNA into lentiCRISPR v2 vector (Addgene #52961; Watertown, MA). sgRNA primers (targeting on Hdac3 Exon2) were 5 ’ -CACCGCATAGCCTAGTCCTGCATTA-3 ’ (forward) and 5 ’ -AAACTAATGCAGGA CTAGGCTATGC-3 ’ (reverse).

Techniques: Quantitative RT-PCR, Western Blot, Mutagenesis

Journal: bioRxiv

Article Title: Epicardial HDAC3 promotes myocardial growth through a novel microRNA pathway

doi: 10.1101/2021.09.16.460538

Figure Lengend Snippet: (A) Volcano plot of miR sequencing of Hdac3 KO and EV MECs. (B) Quantification of Fgf9 and Igf2 expression in MECs after miR mimics treatment (final concentration:10 nM) by qRT-PCR. Gapdh was used as cDNA loading control (* P <0.05 by ANOVA followed by Bonferroni post-hoc test). (C) miR-322 and miR-503 share high similarity of their seed binding motifs to 3 ’ UTRs of Fgf9 and Igf2 . Binding motifs or complementary bases are in red. (D) Quantification of the expression of FGF9 and IGF2 after miR-322 or miR-503 mimic treatment by western blot. SCR: miR scrambles (* P <0.05 by Student ’ s t -test). (E) Quantification of miR-322 and miR-503 in MECs after miR mimic treatment by qRT-PCR. U6 snRNA was used for normalization (* P <0.05 by Student ’ s t -test). (F&G) Quantification of the expression of miR-322 and miR-503 in Hdac3 KO MECs, RGFP966-treated MECs, or E13.5 Hdac3 eko hearts by qRT-PCR (* P <0.05 by Student ’ s t -test).

Article Snippet: To generate stable Hdac3 KO MECs, we cloned Hdac3 sgRNA into lentiCRISPR v2 vector (Addgene #52961; Watertown, MA). sgRNA primers (targeting on Hdac3 Exon2) were 5 ’ -CACCGCATAGCCTAGTCCTGCATTA-3 ’ (forward) and 5 ’ -AAACTAATGCAGGA CTAGGCTATGC-3 ’ (reverse).

Techniques: Sequencing, Expressing, Concentration Assay, Quantitative RT-PCR, Binding Assay, Western Blot

Journal: bioRxiv

Article Title: Epicardial HDAC3 promotes myocardial growth through a novel microRNA pathway

doi: 10.1101/2021.09.16.460538

Figure Lengend Snippet: (A) Quantification of miR-322 and miR-503 after Hdac3 KO or EV MECs were infected with LentimiRa-GFP-miRZip (miR-322 or miR-503) or pGreenPuro Scramble Hairpin control lentivirus (SCR) respectively. miR levels were quantified by qRT-PCR ( n =3, * P <0.05 when compared to the SCR group by ANOVA followed by Bonferroni post-hoc test). (B) Quantification of the expression of FGF9 and IGF2 after miRZip lentiviral treatment by western blot (* P <0.05 when compared to the SCR group by ANOVA followed by Bonferroni post-hoc test).

Article Snippet: To generate stable Hdac3 KO MECs, we cloned Hdac3 sgRNA into lentiCRISPR v2 vector (Addgene #52961; Watertown, MA). sgRNA primers (targeting on Hdac3 Exon2) were 5 ’ -CACCGCATAGCCTAGTCCTGCATTA-3 ’ (forward) and 5 ’ -AAACTAATGCAGGA CTAGGCTATGC-3 ’ (reverse).

Techniques: Infection, Quantitative RT-PCR, Expressing, Western Blot

Journal: bioRxiv

Article Title: Epicardial HDAC3 promotes myocardial growth through a novel microRNA pathway

doi: 10.1101/2021.09.16.460538

Figure Lengend Snippet: (A) Quantification of H3K27Ac in Hdac3 EV and KO MECs by western blot. (B) Schematic diagram of the miR-322/miR-503 locus from UCSC Genome Browser. In the upstream regulatory regions as well as gene bodies, active epigenetic marker H3K27Ac was identified by the ENCODE project. (C) Quantification of H3K27Ac binding affinity in the miR-322/miR-503 promoter region in Hdac3 KO and EV MECs by ChIP-qPCR. Primers targeting a gene desert region were used as a negative control ( n =3, * P <0.05 when compared to the SCR group by ANOVA followed by Bonferroni post-hoc test). (D) Dual luciferase reporter assays on the -1.5 kb miR-322/miR-503 promoter when Hdac3 is either knocked out or inhibited by RGFP966 (final concentration: 10 uM) treatment. The ratio of firefly: Renilla luciferase light units (RLU) was determined 48 hours after transfection ( n =3, * P <0.05 by Student ’ s t -test). (E) The schematics of the working model. In the developing epicardium, HDAC3 represses the expression of miR-322/miR-503 to release their suppression on the expression of FGF9 and IGF2. When Hdac3 is deleted, the expression of miR-322/miR-503 is increased, which subsequently suppresses the expression of FGF9 and IGF2 to a stronger extent, and the decrease of FGF9 and IGF2 leads to ventricular wall hypoplasia.

Article Snippet: To generate stable Hdac3 KO MECs, we cloned Hdac3 sgRNA into lentiCRISPR v2 vector (Addgene #52961; Watertown, MA). sgRNA primers (targeting on Hdac3 Exon2) were 5 ’ -CACCGCATAGCCTAGTCCTGCATTA-3 ’ (forward) and 5 ’ -AAACTAATGCAGGA CTAGGCTATGC-3 ’ (reverse).

Techniques: Western Blot, Marker, Binding Assay, Negative Control, Luciferase, Concentration Assay, Transfection, Expressing

Journal: Investigative ophthalmology & visual science

Article Title: Ecel1 Knockdown With an AAV2-Mediated CRISPR/Cas9 System Promotes Optic Nerve Damage-Induced RGC Death in the Mouse Retina.

doi: 10.1167/iovs.18-23784

Figure Lengend Snippet: FIGURE 2. Optic nerve crush strongly induced Ecel1 expression in the retinas of the mice. (A) qRT-PCR was performed to analyze the Ecel1 mRNA expression level, normalized to Gapdh mRNA (n ¼ 7–11 each group). (B) The protein level of Ecel1 in the retinas was examined with an immunoblot analysis. Beta actin was used as an internal control. (C) Retinal protein was immunolabeled with anti-Ecel1 with or without a blocking peptide. (D) Immunohistochemistry showed that Ecel1 was absent from the retinas of mice that underwent a sham operation, while Ecel1 protein was abundantly expressed in the GCL on day 4 after optic nerve crush. (E) Ecel1 protein was strikingly localized in the mouse GCL on day 4 after optic nerve crush and not in the other cell layers. (F) Histogram showing the number of Ecel1-positive cells in the GCL 4 days after optic nerve crush (n ¼ 4 each group). (G) Immunoreaction for Ecel1 was colocalized with RBPMS, a marker of the RGCs. Error bars denote standard deviation. NC, nerve crush. **P < 0.01, ***P < 0.001. Scale bar: 20 lm.

Article Snippet: To construct adeno-associated virus (AAV) plasmids expressing Staphylococcus aureus Cas9 and single guide RNA sequences under control of the CMV and U6 promoters, respectively, doublestrand DNA oligonucleotides corresponding to the designed guide RNA sequences were inserted into a BsaI recognition site, as follows: pX601-AAV-CMV::NLS-SaCas9-NLS-3xHAbGHpA;U6::BsaI-sgRNA (pX601; a gift from Feng Zhang; Addgene plasmid no. 61591).27 Guide RNA sequences targeting Ecel1 and cyan fluorescent protein (CFP), as a negative control, were used in this study (see Fig. 4A).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Control, Immunolabeling, Blocking Assay, Immunohistochemistry, Marker, Standard Deviation

Journal: Investigative ophthalmology & visual science

Article Title: Ecel1 Knockdown With an AAV2-Mediated CRISPR/Cas9 System Promotes Optic Nerve Damage-Induced RGC Death in the Mouse Retina.

doi: 10.1167/iovs.18-23784

Figure Lengend Snippet: FIGURE 3. Ecel1 expression was strongly induced in damaged RGCs treated with vinblastine but not in those treated with NMDA. Relative expression levels of Thy-1.2 (A), Rbpms (B), and Ecel1 (C) in vinblastine-treated retinas, and Thy1 (D), Rbpms (E), and Ecel1 (F) in NMDA-treated retinas, were determined with quantitative RT-PCR and normalized to the Gapdh level. Error bars denote SD (vinblastine; n¼7–8, NMDA; n¼6–10 for each group at each time point). Vin, vinblastine. *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: To construct adeno-associated virus (AAV) plasmids expressing Staphylococcus aureus Cas9 and single guide RNA sequences under control of the CMV and U6 promoters, respectively, doublestrand DNA oligonucleotides corresponding to the designed guide RNA sequences were inserted into a BsaI recognition site, as follows: pX601-AAV-CMV::NLS-SaCas9-NLS-3xHAbGHpA;U6::BsaI-sgRNA (pX601; a gift from Feng Zhang; Addgene plasmid no. 61591).27 Guide RNA sequences targeting Ecel1 and cyan fluorescent protein (CFP), as a negative control, were used in this study (see Fig. 4A).

Techniques: Expressing, Quantitative RT-PCR

Journal: Investigative ophthalmology & visual science

Article Title: Ecel1 Knockdown With an AAV2-Mediated CRISPR/Cas9 System Promotes Optic Nerve Damage-Induced RGC Death in the Mouse Retina.

doi: 10.1167/iovs.18-23784

Figure Lengend Snippet: FIGURE 4. Ecel1 knockdown with the CRISPR/Cas9 system promoted RGC loss after optic nerve crush. (A) Structure of the mouse Ecel1 gene; the 2nd exon is magnified. The mouse Ecel1 gene has 18 exons and 17 introns. The translation start codon (ATG) is located on the second exon. The guide RNA sequences for Ecel1 and for CFP as a control are shown in the box. (B) T7 Endonuclease 1 (T7E1) assay revealing genome editing at the Ecel1 locus in sorted mCherry-expressing cells from AAV2-mCherry and AAV2-CRISPR/Cas9-injected retinas. Asterisks indicated indel formation. (C) Mutation pattern sequencing of Ecel1 locus. (D) Immunoblotting with an anti-Ecel1 antibody in retinas injected with AAV2-CRISPR/Cas9 4 days after optic nerve crush. (E) Quantification of Ecel1 protein levels normalized with b-actin in (D) (n ¼ 3 each). (F) Immunostaining images for Ecel1 with AAV2-CRISPR/Cas9-Ecel1 treatment 4 days after optic nerve crush. (G) Histogram showing the number of Ecel1-positive cells in the GCL 4 days after optic nerve crush and treatment with AAV2-CRISPR/Cas9. (H) Representative images of FG-labeled RGCs 7 days after optic nerve crush in mice

Article Snippet: To construct adeno-associated virus (AAV) plasmids expressing Staphylococcus aureus Cas9 and single guide RNA sequences under control of the CMV and U6 promoters, respectively, doublestrand DNA oligonucleotides corresponding to the designed guide RNA sequences were inserted into a BsaI recognition site, as follows: pX601-AAV-CMV::NLS-SaCas9-NLS-3xHAbGHpA;U6::BsaI-sgRNA (pX601; a gift from Feng Zhang; Addgene plasmid no. 61591).27 Guide RNA sequences targeting Ecel1 and cyan fluorescent protein (CFP), as a negative control, were used in this study (see Fig. 4A).

Techniques: Knockdown, CRISPR, Control, Expressing, Injection, Mutagenesis, Sequencing, Western Blot, Immunostaining, Labeling