Journal: Developmental cell

Article Title: ESRP1 mutations cause hearing loss due to defects in alternative splicing that disrupt cochlear development

doi: 10.1016/j.devcel.2017.09.026

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Total RNA (200ng) was used for poly A selected RNA-seq library preparation using the NEBNext® Ultra ™ Directional RNA Library Prep Kit from Illumina® (mRNA) (New England Biolabs) (products: NEBNext® Poly(A) mRNA Magnetic Isolation Module (E7490S) and NEBNext® Ultra ™ Directional RNA Library Prep Kit for Illumina® (E7420S).

Techniques: Transduction, Recombinant, Isolation, Derivative Assay, Modification, Sequencing, CRISPR, Software

Journal: Life Science Alliance

Article Title: Combined ADAMTS10 and ADAMTS17 inactivation exacerbates bone shortening and skin phenotypes

doi: 10.26508/lsa.202503232

Figure Lengend Snippet: (A) Domain organization of ADAMTS17 shows location and targeting of exon 3 by CRISPR/Cas9 gRNA to induce nonhomologous end joining. The nucleotide and amino acid sequence of the ADAMTS17 WT allele (green) and after AT insertion (red) are indicated. The dinucleotide insertion induced a frameshift, which resulted in a premature stop codon after 12 amino acids. (B) Sanger sequencing traces of a PCR product generated with primers flanking exon 3 showing the AT insertion (underlined) in the Adamts17 KO. (C) Quantitative real-time PCR using cDNA prepared from WT and Adamts17 KO lung tissue as a template shows significant reduction of ADAMTS17 mRNA in the KO (n = 3). (D) Micrographs of ADAMTS17 immunostaining of sections through WT and Adamts17 KO skin (left), DKO growth plates (middle), and of primary DKO mouse skin fibroblasts (right). The signal in the dermis around hair follicles, in growth plate chondrocytes, and in fibroblasts and their ECM originating from the monoclonal ADAMTS17 antibody was strongly reduced in KO and DKO tissues and cells, indicating lack of ADAMTS17 protein in Adamts17 KO mice. (E) Pie chart showing Mendelian distribution of genotypes recovered from Adamts17 Het intercrosses at the time of genotyping (P7–P10) (n = 94 mice). (F) Breeding scheme to generate WT, Adamts10 KO (10KO), Adamts17 KO (17KO), and DKO mice. (G) Pie chart showing distribution of genotypes recovered from Adamts10 Het; Adamts17 Het intercrosses at P7–P10 (n = 180 mice). Statistical analysis was performed using Chi square calculation. (H) Kaplan–Meier survival analysis of DKO mice. The numbers of observed dead/total mice for the individual genotypes are indicated in brackets. Statistical significance was determined using a log-rank test. (I) Whole mount images of WT, 10KO, 10KO;17Het mice at 4 wk of age show progressive reduction in body size. (J) Bar graphs showing body weights of 4-wk-old mice of the indicated genotypes. The number of mice is indicated below the genotypes. (I, K) Bar graphs showing body weight normalized to average femur length for the genotypes that were significantly different in (I). Bars in (C) indicate mean values and whiskers the SD. In (J, K) floating bars indicate the 25th–75th percentile range, lines the mean value, and whiskers the SD. (C, J, K) Statistical differences in (C) were determined using a two-sided t test and (J, K) were using a one-way ANOVA with post hoc Tukey test. a, P < 0.05 compared with WT.

Article Snippet: Human embryonic kidney (HEK) 293 cells (CRL-1573) and adult human dermal fibroblasts (HDF, PCS-201-012) were purchased from ATCC.

Techniques: CRISPR, Sequencing, Generated, Real-time Polymerase Chain Reaction, Immunostaining

Journal: Life Science Alliance

Article Title: Combined ADAMTS10 and ADAMTS17 inactivation exacerbates bone shortening and skin phenotypes

doi: 10.26508/lsa.202503232

Figure Lengend Snippet: (A) Micrographs of Masson’s trichrome–stained cross sections through dorsal skin from 4-wk-old WT, Adamts10 KO (10KO), Adamts17 KO (17KO), and DKO mice. ED, epidermis; D, dermis; HD, hypodermis; PC, panniculus carnosus. (B, C, D) Bar graphs showing quantification of overall skin thickness (B) and the thicknesses of the epidermis, dermis, hypodermis (C), and panniculus carnosus (p. carnosus, (D)). Individual data points represent multiple measurements along the different skin layers from n = 3 mice/genotype. (E) Stacked bar graphs showing the relative proportions of individual skin layers. The percentage values are indicated. (F) Bar graphs show the quantification of hair follicle numbers in the skin for each genotype. (G, H) Bar graphs showing normalized gene expression in fragments per kilobase of transcript per million mapped reads (FPKM) for Adamts10 and Adamts17 in individual skin cell types at E14.5 (G) and P5 (H). Data were extracted from the Hair-GEL database ( ; ). (I, J, K) Micrographs showing the localization of ADAMTS17 mRNA (red/dark purple) in WT skin cross sections at E13.5 (I), E16.5 (J), and P0 (K) detected by RNAscope in situ hybridization with a probe specific for ADAMTS17 mRNA. Sections were counterstained with hematoxylin. (L) Micrograph of ADAMTS17 immunostaining (green) of cross sections through WT skin. Nuclei were stained with DAPI (blue). (M) Micrographs of primary mouse skin fibroblasts after immunostaining for fibrillin-1 (red) and fibronectin (green). Nuclei were counterstained with DAPI (blue). (M, N) Quantification of mean fluorescence intensity from (M) (n = 4 biological replicates). In (B, C, D, F), floating bars indicate 25th–75th percentile range, lines the mean value and whiskers the SD. In (N), the bars represent the mean value and the whiskers the SD. Statistical differences in (B, C, D, F, N) were determined using a one-way ANOVA with post hoc Tukey test. a, P < 0.05 compared with WT; b, P < 0.05 compared with Adamts10 KO; P < 0.05 compared with Adamts17 KO.

Article Snippet: Human embryonic kidney (HEK) 293 cells (CRL-1573) and adult human dermal fibroblasts (HDF, PCS-201-012) were purchased from ATCC.

Techniques: Staining, Gene Expression, RNAscope, In Situ Hybridization, Immunostaining, Fluorescence

Journal: Life Science Alliance

Article Title: Combined ADAMTS10 and ADAMTS17 inactivation exacerbates bone shortening and skin phenotypes

doi: 10.26508/lsa.202503232

Figure Lengend Snippet: (A) Domain organization of ADAMTS10 and ADAMTS17, which is identical. The degenerate (ADAMTS10) and canonical (ADAMTS17) furin-processing sites and the localization of the catalytic residue Glu-390 in ADAMTS17 (17) that was mutated into Ala to generate proteolytically inactive ADAMTS17-EA (17-EA) are indicated. The domain organization of the catalytic (17-PCD) and ancillary (17-AD) domain constructs is indicated. (B) Schematic representation of experimental design for coculture of human dermal fibroblasts (HDF) with HEK293 cells stably expressing 17- or 17-EA (left) or co-transfection of 17- or 17-EA–encoding plasmids with FN1 or COL6A2-encoding plasmids in HEK293 cells (right). (C) Volcano plot showing N-terminally labeled peptides identified by N-terminomics method TAILS in conditioned medium from ADAMTS17-expressing HEK293 cells cocultured with HDFs. Peptides present only in samples from WT ADAMTS17 (red) or enriched in conditioned medium from WT ADAMTS17 cocultures compared with the cocultures with proteolytically inactive ADAMTS17-EA suggest ADAMTS17 substrates. (D) Venn diagram showing overlap of ADAMTS17-cleaved proteins (TAILS) from coculture systems (left) and binding partners for the ADAMTS17 ancillary domain (17-AD) identified by yeast-2-hybrid screening with a human placenta–derived cDNA library (right). Note that fibronectin (FN1) and COL6 were independently identified in both screens. (E) Domain organization of fibronectin (FN1, NP_997647 ) showing the localization of the domains that interacted with 17-AD (grey box, bolded amino acid sequence) and the localization of the peptide identified by TAILS (red bar, red amino acid sequence). (F) MS2 spectrum of the N-terminally labeled FN1 peptide (GNSVNEGLNQPTDDSCFDPYTVSHYAVGDEWER) showing b- and y ions. (G) Western blot detection of endogenous fibronectin in conditioned medium (Med) and cell lysates (Lys) collected after coculture of 17- or 17-EA–expressing HEKs with HDFs. A monoclonal (green) and four different polyclonal (red) anti-fibronectin antibodies were used. (H) Western blot detection of recombinant fibronectin (rFN) in conditioned medium (Med) and cell lysate (Lys) collected after co-expression of 17 or 17-EA with rFN in HEK293 cells. A polyclonal anti-fibronectin antibody (red) and a monoclonal anti V5-tag antibody (green) were used to detect rFN.

Article Snippet: Human embryonic kidney (HEK) 293 cells (CRL-1573) and adult human dermal fibroblasts (HDF, PCS-201-012) were purchased from ATCC.

Techniques: Residue, Construct, Stable Transfection, Expressing, Cotransfection, Labeling, Binding Assay, Derivative Assay, cDNA Library Assay, Sequencing, Western Blot, Recombinant

Journal: Life Science Alliance

Article Title: Combined ADAMTS10 and ADAMTS17 inactivation exacerbates bone shortening and skin phenotypes

doi: 10.26508/lsa.202503232

Figure Lengend Snippet: (A) Domain organization of COL6A2 (NP_0018403) showing the localization of the domains that interacted with 17-AD (grey box, bolded amino acid sequence). (B) Domain organization of COL6A3 ( NP_004360 ) showing the localization of the peptide identified by MS (red bar, red amino acid sequence). (C) MS2 spectrum of the N-terminally labeled ADAMTS17-digested COL6A3 peptide (SDDEVDDPAVELkQFGVAPF) showing b- and y ions. (D) Western blot of endogenous (end.) COL6 (red) in conditioned medium (Med) and cell lysate (Lys) collected from cocultures of 17- or 17-EA–expressing HEK293 cells with HDFs. (E) Micrographs of endogenous COL6A1 deposition (red) in the ECM of HDFs cocultured with 17- or 17-EA–expressing HEK293 cells. Nuclei were stained with DAPI (blue). (F) Quantification of the mean fluorescence intensity of the COL6A1 signal (n = 3 replicates). (G) Micrographs of endogenous COL6A1 deposition (red) in the ECM of HDF after culture in the presence of conditioned medium from 17- or 17-EA–expressing HEK293 cells. Nuclei were stained with DAPI (blue). (H) Quantification of the mean fluorescence intensity of the COL6A1 signal (n = 3 replicates). (I) Western blot of recombinant COL66A2 (rCOL6) in conditioned medium (Med) and cell lysate (Lys) collected after co-expression of 17 or 17-EA and rCOL6A2 in HEK293 cells using a monoclonal anti FLAG-tag antibody (green). (J) Micrographs of HDFs cultured in the presence of 50 μg/ml of purified recombinant 17-PCD and 17-AD protein (see for domain organization) costained for endogenous COL6A1 (red) and the Myc-tag of the recombinant ADAMTS17 protein fragments (green). Nuclei were stained with DAPI (blue). (K) Micrographs of adult HDFs and Weill–Marchesani syndrome (WMS) patient–derived dermal fibroblasts (WMS-DF) for endogenous COL6A1 (red). Nuclei were counterstained with DAPI (blue). (L) Quantification of the mean fluorescence intensity of the COL6A1 signal (n = 3 replicates, 2–3 fields of view). (M) Western blot of endogenous COL6A1 (red) and GAPDH (green) in conditioned medium (Med) and cell lysate (Lys) collected from HDF and WMS-DF cultures. (N) Quantification of COL6A1 band mean fluorescence intensities normalized to GAPDH. In (E, G, M), bars represent the mean value and whiskers the SD. In (K), the floating bars indicate the 25th–75th percentile range, the lines the mean value, and whiskers the SD. Statistical differences in (E, G, K, M) were determined using a two-sided t test.

Article Snippet: Human embryonic kidney (HEK) 293 cells (CRL-1573) and adult human dermal fibroblasts (HDF, PCS-201-012) were purchased from ATCC.

Techniques: Sequencing, Labeling, Western Blot, Expressing, Staining, Fluorescence, Recombinant, FLAG-tag, Cell Culture, Purification, Derivative Assay

Journal: Science advances

Article Title: Oncogenic transcription factors instruct promoter-enhancer hubs in individual triple negative breast cancer cells.

doi: 10.1126/sciadv.adl4043

Figure Lengend Snippet: Fig. 3. Enhancer activity compacts SOX9 promoter-enhancer hub in individual TNBC cells. (A and B) SOX9 promoter participates in multiway interactions with its distal enhancer clusters in individual TNBC MB157 cells. Left: Allele percentages with SOX9 promoter interacting with SOX9.EC1, SOX9.EC3, or both (A) and SOX9.EC2, SOX9. EC3, or both (B) in MB157 (n = alleles). Right-top: SOX9 locus schematic, three-color DNA FISH 50-kb probes at SOX9 promoter (green), SOX9.EC3 (magenta), and SOX9.EC1 (A, red) or SOX9.EC2 (B, yellow). Locations per fig. S3A. Right-bottom: Representative cells. Blue: 4′,6-Diamidino-2-phenylindole (DAPI). (C and F) SOX9 enhancers inactiva- tion expands SOX9-EC1-EC3 and SOX9-EC2-EC3 hubs in individual TNBC MB157 cells. Cumulative distribution functions (CDFs) of SOX9-EC1-EC3 (C) and SOX9-EC2-EC3 (F) spatial perimeters in each MB157-dCas9-KRAB expressing control (CTRL), SOX9.EC1, SOX9.EC2, or SOX9.EC3 sgRNA [Kolmogorov-Smirnov (KS) test, n = cells]. Mean (±SD) perimeters (micrometers): (C) Left: CTRL/SOX9.EC1 sgRNA: 3.78 (±2.63)/4.36 (±2.63); middle: CTRL/SOX9.EC2 sgRNA: 3.78 (±2.63)/4.47 (±2.64); right: CTRL/SOX9.EC3 sgRNA: 3.39 (±2.56)/4.28 (±2.65). (G) Left: CTRL/SOX9.EC1 sgRNA: 3.83 (±2.71)/4.45 (±2.72); middle: CTRL/SOX9.EC2 sgRNA: 3.19 (±2.44)/4.26 (±2.61); right: CTRL/SOX9.EC3 sgRNA: 3.83 (±2.71)/4.22 (±2.56). (D and G) Allele percentages with SOX9 promoter interacting with SOX9.EC1, SOX9.EC3, or both (D) and SOX9.EC1, SOX9. EC3, or both (G) in MB157-dCas9-KRAB expressing CTRL, SOX9.EC1, SOX9.EC2, or SOX9.EC3 sgRNA (n = alleles). (E and H) Representative cells of 3C and 3D (E) or 3F and 3G (H). Blue: DAPI. (I) SOX9 promoter inactivation decreases SOX9-EC1-EC3 three-way interaction frequency across individual alleles in TNBC MB157. Top-left: Allele percent- ages with SOX9 promoter interacting with SOX9.EC1, SOX9.EC3, or both in MB157-dCas9-KRAB expressing CTRL or SOX9 promoter sgRNA (SOX9.P sgRNA) (n = alleles). Bottom-left: CDFs of SOX9-EC1-EC3 spatial perimeter in each MB157-dCas9-KRAB cell (KS test, n = cells). CTRL/SOX9.P sgRNA mean (±SD) perimeter: 3.90 (±2.62)/4.44 (±2.66) μm. Right: Representative cells. Blue: DAPI. Scale bars, 3 μm for nuclei and 0.5 μm for alleles.

Article Snippet: For the transcription factor CRISPR screen sgRNA pooled library, lentivirus was produced by transfecting HEK293T cells with helper plasmids (VSVG and psPAX2; Addgene: #12260) using FuGene HD (Promega, catalog no. E2311).

Techniques: Activity Assay, Expressing, Control

Journal: Science advances

Article Title: Oncogenic transcription factors instruct promoter-enhancer hubs in individual triple negative breast cancer cells.

doi: 10.1126/sciadv.adl4043

Figure Lengend Snippet: Fig. 6. SOX9 regulates oncogene MYC by positioning its enhancers. (A) Genome tracks showing enrichment of pairwise MYC enhancer-enhancer and promoter- enhancer interactions in a population of MB157 cells. From top to bottom: Colored circles marking location of Oligopaint DNA FISH probes labeling 50-kb regions at MYC promoter (green), MYC.EC1 (magenta), MYC.EC2 (red), MYC.EC3 (yellow), and T-ALL-restricted enhancer (black), H3K27ac and SOX9 levels as measured by ChIP-seq, and normalized interaction frequency as measured by SMC1 HiChIP at the MYC locus in MB157. MYC enhancer clusters are marked by grey boxes. (B) A total of 80% of differ- entially expressed genes with SOX9-bound promoter and distal enhancer participate in ensemble hyper-interacting hubs. MB157 hubs plotted in ascending order of their total connectivity as measured by SMC1 HiChIP in TNBC MB157. Hyper-interacting promoter-enhancer hubs are defined as the ones above the elbow of the ranked total connectivity plot. Hyper-interacting ensemble promoter-enhancer hubs containing genes that are significantly down-regulated in MB157-Cas9 cells transfected with SOX9 targeting sgRNA versus control sgRNA for 4 days and have SOX9-bound promoter and distal enhancer are marked in orange. (C to E) SOX9 loss significantly in- creases 3D distances between the MYC promoter and SOX9-bound MYC.EC2 (C) or MYC.EC3 (D) and SOX9-unbound MYC.EC1 (E) in individual cells. CDFs (left) and box and whiskers (middle) of the distances between the MYC promoter and SOX9-bound MYC.EC2 (C) and MYC.EC3 (D) and SOX9-unbound MYC.EC1 (E) in each MB157-Cas9 6 days after transduction with control sgRNA (CTRL) or SOX9-targeting sgRNA (SOX9 KO) (KS test, n = cells). Probe locations per 6A. CTRL/SOX9 KO mean (±SD) distance between MYC promoter and MYC.EC2: 0.389 (±0.358)/0.749 (±0.666) μm; MYC.EC3: 0.447 (±0.457)/0.591 (±0.551) μm; MYC.EC1: 0.494 (±0.447)/0.651 (±0.556) μm. Right: Represen- tative cells. Scale bar per 3A. Blue: DAPI.

Article Snippet: For the transcription factor CRISPR screen sgRNA pooled library, lentivirus was produced by transfecting HEK293T cells with helper plasmids (VSVG and psPAX2; Addgene: #12260) using FuGene HD (Promega, catalog no. E2311).

Techniques: Labeling, ChIP-sequencing, HiChIP, Transfection, Control, Transduction

Journal: Science advances

Article Title: Oncogenic transcription factors instruct promoter-enhancer hubs in individual triple negative breast cancer cells.

doi: 10.1126/sciadv.adl4043

Figure Lengend Snippet: Fig. 7. SOX9 loss decompacts MYC promoter-enhancer hubs. (A) MYC promoter participates in multiway interactions with its distal enhancer clusters in individual TNBC MB157 and MDA-MB-468 but not ER+ MCF7. Left: Percentage of alleles with MYC promoter interacting (<350 nm) with SOX9-unbound MYC.EC1, SOX9-bound MYC.EC3, or both MYC.EC1 and MYC.EC3 in MB157, MDA-MB-468, and MCF7 as measured by three-color Oligopaint DNA FISH with probes marked in Fig. 6A top genome track (n = alleles). Right: Representative MB157, MDA-MB-468, and MCF7 nuclei and two magnified alleles from three-color DNA FISH. Scale bar per 3A. Blue: DAPI. (B and C) SOX9 loss expands MYC-EC1-EC2 (B) and MYC-EC1-EC3 (C) promoter-enhancer hubs in individual MB157 and decreases three-way interaction frequency across individual alleles. Left: CDFs of MYC-EC1-EC2 (B) and MYC-EC1-EC3 (C) spatial perimeters in each MB157-Cas9 cell expressing CTRL or SOX9 KO sgRNA (KS test, n = cells). Probe locations per 6A. CTRL/SOX9 KO mean (±SD) perimeters MYC-EC1-EC2 (B): 3.84 (±2.70)/4.53 (±2.67) μm; MYC-EC1-EC3 (C): 3.10 (±2.58)/3.90 (±2.64) μm (n = cells). Middle: Allele per- centages with MYC promoter interacting (<350 nm) with MYC.EC1, MYC.EC2, or both MYC.EC1 and MYC.EC2 (B) and MYC.EC1, MYC.EC3, or both MYC.EC1 and MYC.EC3 (C) in CTRL and SOX9 KO MB157-Cas9. Right: Representative cells. Scale bar per 3A. Blue: DAPI.

Article Snippet: For the transcription factor CRISPR screen sgRNA pooled library, lentivirus was produced by transfecting HEK293T cells with helper plasmids (VSVG and psPAX2; Addgene: #12260) using FuGene HD (Promega, catalog no. E2311).

Techniques: Expressing

Journal: bioRxiv

Article Title: CFIm25 regulates human stem cell function independently of its role in mRNA alternative polyadenylation

doi: 10.1101/2021.12.08.471721

Figure Lengend Snippet: (A) SYBR Green staining of PCR product and DNA fragments resulted from T7 Endonuclease I assay. PCR product amplified from CFIm25 gene locus is indicated by the arrow and digested DNA fragments is indicated by the bracket. Two representative replicates experiments are shown. (B) Sanger sequencing of CFIm25 gene locus to confirm the genomic mutations/deletions in three CRISPR-Cas9 system-mediated H9 cell clones. Start codon ‘ATG’ is colored green, nucleotides colored in red represent mutations, and symbol “-” stands for nucleotide deletion at the corresponding position. Genomic positions targeted by gRNAs are underlined. (C) Cell proliferation rate measurement by CCK-8 kit in mock and three CFIm25-mutant H9 cell lines. The starting cell density in this experiment is 7500 cell per well of 96 well plates. Three independent experiments have been carried out and representative results are shown. (D) Quantifications of the percentages of cells at different stages during cell cycles. The results are from three independent experiments. A representative result is shown in (m: mutant; OE: overexpression). Student’s t-test was used to estimate the significance of the change. *P<0.05; n.s.: non-significant. (E) RT-qPCR analysis of the expression level of four pluripotency-associated markers in mock and CFIm25-m hESCs. (F) Immunostaining analysis of pluripotency marker OCT4 in mock and CFIm25-m hESCs. (G) Phase-contrast images of mock and CFIm25-m hESC clones.

Article Snippet: These results are in line with the observation that the molecular weight of the band in mutant cells is slightly smaller than that in control cells using the CFIm25 antibody from Proteintech ( ; Figure 1-source data file 3).

Techniques: SYBR Green Assay, Staining, T7EI Assay, Amplification, Sequencing, CRISPR, Clone Assay, CCK-8 Assay, Mutagenesis, Over Expression, Quantitative RT-PCR, Expressing, Immunostaining, Marker

Journal: bioRxiv

Article Title: CFIm25 regulates human stem cell function independently of its role in mRNA alternative polyadenylation

doi: 10.1101/2021.12.08.471721

Figure Lengend Snippet: CFIm25 knockdown/mutation impacts the cell proliferation rate and differentiation potential of H9 cell line. (A) Western blot analysis of CFIm25 and CFIm59/68 proteins in cell lysates prepared from two controls and three CFIm25 gene-edited H9 cell lines. GAPDH serves as sample loading control (Control: H9 cells; Mock control: eCRISPR empty vector-transfected H9 cells; CFIm25 m1-3: eCRISPR-CFIm25 gRNAs transfected H9 cells). The primary antibody 1 against CFIm25 is from Santa Cruz Company, and antibody 2 is from Proteintech. (B) Western blot analysis of CFIm25 and indicated protein (peptide) in cell lysates prepared from Mock, CFIm25-m, and CFIm25-m plus 3XFIag-CFIm25 overexpression cells. At least three independent experiments have been carried out and representative images are shown. The primary antibody for CFIm25 is from santa cruz (sc-81109). (C) Cell proliferation rate measurement by CCK-8 kit for indicated cell lines. The growth rate of hESCs is largely dependent on starting cell density. The starting cell density in this experiment is 5000 per well of 96 well plates. Three independent experiments have been carried out and representative results are shown. (D) Flow Cytometry analysis of cell cycle using a Propidium Iodide Flow Cytometry Kit in the indicated cell lines (m: mutant; OE:overexpression). The right panel shows the representative result of the percentages of cells during different stages of cell cycle. The quantification of three independent experiments is shown in . (E) RT-qPCR analysis of the expression level of corresponding lineage differentiation markers in indicated cell lines during trilineage differentiation. Three independent experiments have been carried out and quantified. Student’s t-test was used to estimate the significance of the change. *p<0.05. ns: non-significant. (F) Quantification of the yield of cardiomyocytes by performing fluorescence-activated cell sorting (FACS) analysis in the indicated cell lines during cardiomyocytes differentiation from three independent experiments. cTnT antibodies were used in FACS experiment. Bottom panel is the quantification from three representative experiments (m: mutant; OE: overexpression). Student’s t-test was used to estimate the significance: * p < 0.05.

Article Snippet: These results are in line with the observation that the molecular weight of the band in mutant cells is slightly smaller than that in control cells using the CFIm25 antibody from Proteintech ( ; Figure 1-source data file 3).

Techniques: Knockdown, Mutagenesis, Western Blot, Control, Plasmid Preparation, Transfection, Over Expression, CCK-8 Assay, Flow Cytometry, Quantitative RT-PCR, Expressing, Fluorescence, FACS

Journal: bioRxiv

Article Title: CFIm25 regulates human stem cell function independently of its role in mRNA alternative polyadenylation

doi: 10.1101/2021.12.08.471721

Figure Lengend Snippet: Effect of CFIm25 knockdown/mutation on the global mRNA alternative polyadenylation (APA) profile and expression level of poly(A+) transcripts in H9 cell line. (A) mRNA alternative polyadenylation (APA) change in mock and CFIm25-m(1-3) H9 cell lines. 3’-seq analysis of APA in mock and CFIm25-m H9 cells, Log2(proximal/distal ratio) are plotted for mock (y-axis) and CFIm25-m H9 cells (x-axis). Statistically significant changes are highlighted in blue (distal to proximal shift) and red (proximal to distal shift). The numbers of APA changes are shown in the column graph. (B) Volcano plot showing the expression level change of poly(A+) mRNA in mock and CFIm25-m (1-3) H9 cells. Significant changes (p<0.05, fold change>l) were colored red (up-regulated in CFIm25-m cells in comparison to mock cells) or green (down-regulated in CFIm25-m cells in comparison to mock cells), blue dots shows the changes either not statistically significant (p>0.05) or less reliable (fold change<1). Genes for subsequent studies are circled. (C) IGV track screen shots showing mRNA-seq and 3’-seq results for rex1 gene in mock and CFIm25-m H9 cells. (D) Gene ontology analysis of the group of down-regulated genes (277 genes) upon CFIm25-m using the Gene Ontology Consortium platform ( http://geneontology.org/ ). Gene ontology terms (y axis) and corresponding p-values (x axis) are shown. (E) RT-qPCR analysis of the expression level of indicated genes in mock and CFIm25-m (1-3) H9 cells. The results of three independent experiments have been quantified. Student’s t-test was used to estimate the significance of the change. *P<0.05.

Article Snippet: These results are in line with the observation that the molecular weight of the band in mutant cells is slightly smaller than that in control cells using the CFIm25 antibody from Proteintech ( ; Figure 1-source data file 3).

Techniques: Knockdown, Mutagenesis, Expressing, Comparison, Quantitative RT-PCR

Journal: bioRxiv

Article Title: CFIm25 regulates human stem cell function independently of its role in mRNA alternative polyadenylation

doi: 10.1101/2021.12.08.471721

Figure Lengend Snippet: (A) IGV track screen shots showing the 3’-seq results for dicer1 and ccnd1 genes in mock and CFIm25-m H9 cells. Two predominant PASs within 3’UTR regions are indicated with arrows. Proximal or distal PAS are named according to their positions relative to gene 5’ end. (B) Measurement of canonical SVL PAS processing efficiency using pPASPORT system in mock and CFIm25-m cells, SVL-m (AAUAAA core hexamer was replaced as AACAAA) serves as negative control. SVL or SVL(m) PAS were inserted into multiple cloning sites between Renilla luciferase (Rlu) gene and IRES (internal ribosome entry site). Downstream of the IRES is the Firefly luciferase (Flu) gene. Relative PAS processing efficiency was quantified by calculating the Rlu/Flu ratio. Results from three independent experiments are quantified and represented. Student’s t-test was used to estimate the significance: ns: non-significant. (C) Schematic representation of the SVL RNA substrates used in the biotin–streptavidin pull-down assay (top). The AAUAAA hexamer in wild-type RNA substrate and AACAAA in mutant substrate (boxes) are shown. The asterisk is used to highlight the single nucleotide change. Bottom panel shows the western blot results of known core 3’ processing factors in the RNA-biotin based pull-down experiment using nuclear extracts (NEs) prepared from mock and CFIm25-m H9 cells. Two independent experiments have been carried out and representative results are shown. 5% of the lysate was kept as input. The primary antibody for CFIm25 is from santa cruz (sc-81109). (D) Comparison of gene expression profiling by 3’-seq and mRNA-seq. X axis: total read count for each gene in CFIm25-m sample based on mRNA-seq data; Y axis: total read count for each gene in CFIm25-m based on mRNA-seq results of control and 3’-seq analysis. The mRNA-seq read count for a gene in CFIm25-m sample=(mRNA-seq read count for this gene in control H9) x (expression fold change for this gene based on 3’-seq analyses: CFIm25-m/control). Both X axis and Y axis are in log scale. Pearson’s r=0.65. (E) IGV track screen shots showing mRNA-seq and 3’-seq results for chchd2 and znf717 genes in mock and CFIm25-m H9 cells. (F) Venn diagram showing the number of overlapping and non-overlapping genes that display expression level change upon CFIm25 (m+OE) (blue) and CFIm25-m (yellow) (m: mutant; OE: overexpression). (G) Comparison of the expression level of indicated genes in mock (m+OE) and CFIm25 (m+OE) H9 cells. Data comes from RNA-seq analysis listed in Supplemental Table 4. Student’s t-test was used to estimate the significance: *p<0.05; ns: non-significant.

Article Snippet: These results are in line with the observation that the molecular weight of the band in mutant cells is slightly smaller than that in control cells using the CFIm25 antibody from Proteintech ( ; Figure 1-source data file 3).

Techniques: Negative Control, Cloning, Luciferase, Pull Down Assay, Mutagenesis, Western Blot, Comparison, Gene Expression, Control, Expressing, Over Expression, RNA Sequencing

Journal: bioRxiv

Article Title: CFIm25 regulates human stem cell function independently of its role in mRNA alternative polyadenylation

doi: 10.1101/2021.12.08.471721

Figure Lengend Snippet: CFIm25 knockdown/mutation impacts rex1 gene transcription in H9 cells. (A-B) A pair of primers (F1/R1) was designed to detect the gene transcription readthrough beyond rex1 PAS. The relative expression of extended transcript in mock and CFIm25-m H9 cells was estimated by RT-qPCR analysis shown in . Gapdh gene expression serves as internal control. Student’s t-test was used to estimate the significance of the change. *p<0.05. (C) RT-qPCR analysis of the expression level of rex1 pre-mRNA and mRNA using indicated primers in mock and CFIm25-m cells. Student’s t-test was used to estimate the significance of the change. *P<0.05. (D) Outline of the 3C procedure used to detect chromatin interactions between promoter and terminator region for rex1 gene. (E) PCR product resulting from 3C library amplification using the primers located in the promoter and terminator regions. PCR product targeting gapdh gene serves as input. Cells used for 3C library preparation are indicated above the gel image. (F) Sanger sequencing shows that the PCR products correspond to the ligated DNA fragments of the two regions located at the promoter and terminator of rex1.

Article Snippet: These results are in line with the observation that the molecular weight of the band in mutant cells is slightly smaller than that in control cells using the CFIm25 antibody from Proteintech ( ; Figure 1-source data file 3).

Techniques: Knockdown, Mutagenesis, Expressing, Quantitative RT-PCR, Gene Expression, Control, Library Amplification, Sequencing

Journal: bioRxiv

Article Title: CFIm25 regulates human stem cell function independently of its role in mRNA alternative polyadenylation

doi: 10.1101/2021.12.08.471721

Figure Lengend Snippet: (A) Mock and CFIm25-m (1-3) H9 cells were subjected to Actinomycin D treatment and total RNAs were extracted at the indicated time. RT-qPCR was used to calculate the percentage of the rex1 mRNA left, 28s rRNA serves as normalization control. (B-C) RT-qPCR analysis of rex1 pre-mRNA abundance using chromatin-associated RNAs (B) and BrU-incorporated pre-mRNAs (C) in mock and CFIm25-m H9 cells. gapdh gene product serves as internal normalization control. (D) Comparison of rex1 gene promoter activity in control and CFIm25-m H9 cells using pGL3-basic reporter system. Student’s t-test was used to estimate the significance of the change. ns: non-significant. (E) ChIP-qPCR analysis using primary antibody against CFIm25 and indicated primers targeting different position of rex1 gene locus. For the bar graph, y axis represents the fold change of ChIP signal in mock H9 cells in comparison to that of CFIm25-m cells, x axis stands for the indicated positions across rex1 gene locus. (F) RT-qPCR analysis of rex1 gene expression in undifferentiated H9 cells, and trilineage differentiated cells. Gapdh mRNA was assayed as normalization control. Student’s t-test was used to estimate the significance of the change. *P<0.05.

Article Snippet: These results are in line with the observation that the molecular weight of the band in mutant cells is slightly smaller than that in control cells using the CFIm25 antibody from Proteintech ( ; Figure 1-source data file 3).

Techniques: Quantitative RT-PCR, Control, Comparison, Activity Assay, ChIP-qPCR, Gene Expression

Journal: bioRxiv

Article Title: CFIm25 regulates human stem cell function independently of its role in mRNA alternative polyadenylation

doi: 10.1101/2021.12.08.471721

Figure Lengend Snippet: CFIm25 knockdown/mutation globally impacts transcription dynamics in H9 cells. (A) (left panel) Metagene plots of RNAPII ChIP-seq reads in mock and CFIm25-m H9 cells for actively transcribed genes (FPKM>1 based on mRNA-seq, 14274 genes in total), and its corresponding cumulative frequency plot (right panel). K-S test was used to examine the significance of the difference between the two plots. (B) (left panel) Pie plot showing the genomic annotations of 4024 sites that displayed differential RNAPII binding. Peak calling was performed using MACS2 software and DiffBind package was used to identify the differential binding events. (right panel) Venn diagram showing the numbers of overlapping and non-overlapping genes that displayed differential RNAPII binding and mRNA expression level change. (C) Comparison of RNAPII ChIP-seq and ChIP-qPCR results in mock and CFIm25-m H9 cells for the tested genomic sites. Y axis represents the average fold changes from replicates (ChIP-seq: two replicates; ChIP-qPCR: three replicates). (D) Metagene plots of RNAPII Ser5 ChIP-seq and RNAPII Ser2 ChIP-seq reads for actively transcribed genes in mock and CFIm25-m H9 cells. K-S test was used to examine the significance of the difference between the two plots. (E) Metagene plots of CFIm25 CUT-Tag (top), CFIm59 ChIP-seq (middle), and CFIm68 ChIP-seq (bottom) reads for actively expressed genes in mock and CFIm25-m H9 cells. CFIm25 CUT-Taq profile in CFIm25-m cells was used as normalization control. For CFIm59 and CFIm68 ChIP-seq, K-S test was used to examine the significance of the difference between the two plots. (F) Nuclear run-on assay on the nascent ccdc152 transcript. The gene structure and the primer positions are indicated on the top. The diagram for the nuclear run-on assay is shown in the middle. A representative set of RT-PCR data are shown in the bottom panel. Left gel image: nuclear run on assays followed RT-PCR using primers targeting P1-P5 region. ‘CFIm25-’ represents CFIm25-m cell nuclei, whereas ‘CFIm25 +’ represents mock cell nuclei. Right Bar graph represents RT-qPCR data from three independent experiments. U1 snRNA was assayed as normalization control. Student’s t-test was used to estimate the significance of the change. *P<0.05.

Article Snippet: These results are in line with the observation that the molecular weight of the band in mutant cells is slightly smaller than that in control cells using the CFIm25 antibody from Proteintech ( ; Figure 1-source data file 3).

Techniques: Knockdown, Mutagenesis, ChIP-sequencing, Binding Assay, Software, Expressing, Comparison, ChIP-qPCR, Control, Nuclear Run-on Assay, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR

Journal: bioRxiv

Article Title: CFIm25 regulates human stem cell function independently of its role in mRNA alternative polyadenylation

doi: 10.1101/2021.12.08.471721

Figure Lengend Snippet: (A) Metagene plots of RNAPII ChIP-seq reads for highly expressed genes (top 2000 genes based on mRNA-seq FPKM value), lowly expressed gene (the rest of the genes), 277 down-regulated genes and 310 up-regulated genes upon CFIm25 gene editing. K-S test was used to examine the significance of the difference between the two plots. (B) IGV track screen shot showing RNAPII ChIP-seq result for gapdh gene in mock and CFIm25-m H9 cells. (C) Metagene plots of RNAPII ChIP-seq reads for actively expressed genes in mock and rex1 RNAi H9 cells. K-S test was used to examine the significance of the difference between the two plots. The rex1 gene knockdown efficiency was estimated by RT-qPCR and western blot analysis. Student’s t-test was used to estimate the significance of the change. *P<0.05. (D) Venn diagram showing the number of genes that displayed differential RNAPII/Ser5/Ser2 binding upon CFIm25 depletion. (E) IGV track screen shots showing RNAPII, RNAPII Ser5 and Ser2 ChIP-seq results for dctn5/ccdc152 gene in mock and CFIm25-m H9 cells. (F) Plots showing the normalized CFIm25 CUT&Tag signals in the specific group of genes (up-regulated or down-regulated genes) or individual gene (ccdcl52, dctn5). (G) Nuclear run-on assay on the nascent dctn5 transcript. The gene structure and the probe positions are indicated on the top. The diagram for the nuclear run-on assay is shown in the . A representative set of RT-PCR data are shown in the middle panel. RT-PCR products from mock and CFIm25-m H9 cells are indicated below each set. ‘CFIm25-’ represents CFIm25-m cell nuclei, whereas ‘CFIm25 +’ represents mock cell nuclei. Bar graph represents RT-qPCR data from three independent experiments. U1 snRNA was assayed as normalization control. Student’s t-test was used to estimate the significance of the change. *P<0.05. (H) Comparison of global mRNA APA (left) and gene expression profiles (middle) in control and CFIm25 RNAi cells using the previously reported dataset. The plots are similar to that in . Venn diagram shows that the two groups of genes showing changes do not overlap extensively.

Article Snippet: These results are in line with the observation that the molecular weight of the band in mutant cells is slightly smaller than that in control cells using the CFIm25 antibody from Proteintech ( ; Figure 1-source data file 3).

Techniques: ChIP-sequencing, Knockdown, Quantitative RT-PCR, Western Blot, Binding Assay, Nuclear Run-on Assay, Reverse Transcription Polymerase Chain Reaction, Control, Comparison, Gene Expression

Journal: bioRxiv

Article Title: CFIm25 regulates human stem cell function independently of its role in mRNA alternative polyadenylation

doi: 10.1101/2021.12.08.471721

Figure Lengend Snippet: CFIm25 interacts with LEO1 and impacts the DNA genomic binding profile of LEO1. (A) Western blot analysis of the abundance of indicated proteins in Flag-IPed sample. Flag-IP was performed using extracts from CFIm25-m3 and CFIm25 (m3+3XFIag-CFIm25 overexpression) H9 cells. Input: 1% of the lysates for IP. The primary antibody for CFIm25 is from santa cruz (sc-81109). (B) Schematic representation of full length human LEO1 protein and truncation fragements (F1-F6) (upper). Truncation fragments were fused to pET-28a vector (BamHI and XhoI) for recombinant His-tag protein expression. At least three independent experiments have been performed, and a representative western blotting result of GST-pull down assay using anti-His antibody is shown in the middle picture. As the bait protein, recombinant GST-CFIm25 was stained with Colloidal Coomassie G-250 (Bottom). The percentage of input is indicated in the bracket. (C) Schematic representation of human CFIm25 protein and its N-terminus deletion/mutation derivatives m1-12d/m2-17d/m3-13d represent the 12/17/13 amino acids deletion/mutation proteins produced in CFIm25-mutant (m1-m3) ceils respectively) (upper). Full length and mutant CFIm25 proteins were fused to pGEX-4T3 vector (BamHI and XhoI) for recombinant GST-tag protein expression. In the GST pull-down assay, recombinant His-tag LEO1-F6 protein was used as the prey protein. At least three independent experiments have been performed, and a representative western blotting result of GST-pull down assay using anti-His antibody is shown in the middle picture. GST-fused bait proteins are stained with Colloidal Coomassie G-250 (Bottom). The percentage of input is indicated in the bracket. (D) Metagene plots of LEO1 ChIP-seq and HNRNPL ChIP-seq reads for actively expressed genes in mock and CFIm25-m cells. K-S test was used to examine the significance of the difference between the two plots.

Article Snippet: These results are in line with the observation that the molecular weight of the band in mutant cells is slightly smaller than that in control cells using the CFIm25 antibody from Proteintech ( ; Figure 1-source data file 3).

Techniques: Binding Assay, Western Blot, Over Expression, Plasmid Preparation, Recombinant, Expressing, Pull Down Assay, Staining, Mutagenesis, Produced, ChIP-sequencing

Journal: bioRxiv

Article Title: CFIm25 regulates human stem cell function independently of its role in mRNA alternative polyadenylation

doi: 10.1101/2021.12.08.471721

Figure Lengend Snippet: (A) Western blot analysis of the abundance of LEO1 protein in Flag-IPed sample. Flag-IP was performed using extracts from CFIm25-m3 and CFIm25 (m3+3XFIag-CFIm25 overexpression) H9 cells in the presence of 5 ug/ml RNAse A. Input: 1% of the lysates for IP. (B) Commassie blue staining of purified GST-CFIm25, His-LEO1 (truncation fragments 1-6) fusion proteins (left), and three GST-CFIm25 mutants (the N terminus mutations are based on sequences listed in ). (C) Metagene plots of LEO1 ChIP-seq reads for highly expressed genes (top 2000 genes based on mRNA-seq FPKM value), lowly expressed gene (the rest of the genes), and 277 down-regulated genes upon CFIm25 gene editing. K-S test was used to examine the significance of the difference between the two plots. (D) IGV track screen shot showing LEO1 ChIP-seq result for gapdh gene in mock and CFIm25-m H9 cells.

Article Snippet: These results are in line with the observation that the molecular weight of the band in mutant cells is slightly smaller than that in control cells using the CFIm25 antibody from Proteintech ( ; Figure 1-source data file 3).

Techniques: Western Blot, Over Expression, Staining, Purification, ChIP-sequencing

Journal: bioRxiv

Article Title: CFIm25 regulates human stem cell function independently of its role in mRNA alternative polyadenylation

doi: 10.1101/2021.12.08.471721

Figure Lengend Snippet: CFIm25 targets play roles in hESCs cell proliferation and pluripotency. (A-B; D) RT-qPCR analysis of the expression level of 6 endoderm lineage differentiation markers in indicated cell lines during endoderm lineage differentiation (OE: overexpression; ASO: antisense oligo). Three independent experiments have been performed and quantified results are shown. Student’s t-test was used to estimate the significance of the change. *P<0.05; ns: non-significant. (C) Cell proliferation rate measurement by CCK-8 kit for mock and tusc1 gene overexpression H9 cell lines. Three independent experiments have been performed and quantified results are shown. (E) A schematic model summarizing the key finding in this study. Mutations in human CFIm25 protein N-terminus did not significantly affect cellular mRNA alternative polyadenylation profile, but rather affected transcription process, thereby decreasing the expression level of a group of transcripts associated with pluripotency (such as rex1 gene) and cell proliferation (such as tusc1 gene). CFIm25 depletion/mutation predominantly caused defects in the endoderm/mesoderm differentiation and accelerated the rate of cell growth in H9 cells.

Article Snippet: These results are in line with the observation that the molecular weight of the band in mutant cells is slightly smaller than that in control cells using the CFIm25 antibody from Proteintech ( ; Figure 1-source data file 3).

Techniques: Quantitative RT-PCR, Expressing, Over Expression, CCK-8 Assay, Mutagenesis

Journal: bioRxiv

Article Title: CFIm25 regulates human stem cell function independently of its role in mRNA alternative polyadenylation

doi: 10.1101/2021.12.08.471721

Figure Lengend Snippet: (A) RT-qPCR analysis of the expression level of four pluripotency-associated markers in indicated cell lines. Student’s t-test was used to estimate the significance of the change. *P<0.05; ns: non-significant. (B) Representative phase-contrast images of indicated cell lines. (C) Bar graph showing the percentages of poly(A+) RNAs among total RNAs in mock and CFIm25-m H9 cells. Poly (A+) RNAs were purified by OligodT magnetic beads from total RNAs. Quantification was performed with three independent experiments. Student’s t-test was used to estimate the significance of the change. ns: non-significant.

Article Snippet: These results are in line with the observation that the molecular weight of the band in mutant cells is slightly smaller than that in control cells using the CFIm25 antibody from Proteintech ( ; Figure 1-source data file 3).

Techniques: Quantitative RT-PCR, Expressing, Purification, Magnetic Beads

Journal: eLife

Article Title: A missense in HSF2BP causing primary ovarian insufficiency affects meiotic recombination by its novel interactor C19ORF57/BRME1

doi: 10.7554/eLife.56996

Figure Lengend Snippet:

Article Snippet: Commercial assay or kit , GammaBind G Sepharose , GE Healthcare , 17-0885-02 , Materials and methods section.

Techniques: Recombinant, Generated, Sequencing, CRISPR, DNA Binding Assay, In Situ, cDNA Library Assay, Staining, Plasmid Preparation

Journal: Developmental cell

Article Title: AAGAB controls AP2 adaptor assembly in clathrin-mediated endocytosis

doi: 10.1016/j.devcel.2019.06.013

Figure Lengend Snippet: KEY RESOURCE TABLE

Article Snippet: 293T cells , ATCC , CRL-3216.

Techniques: Virus, Recombinant, Electron Microscopy, Protease Inhibitor, DNA Extraction, Mutagenesis, Sequencing, Library Amplification, CRISPR, Plasmid Preparation, Software

Journal: Cell Stem Cell

Article Title: Loss of Extreme Long-Range Enhancers in Human Neural Crest Drives a Craniofacial Disorder

doi: 10.1016/j.stem.2020.09.001

Figure Lengend Snippet:

Article Snippet: Antibodies used include TWIST1 (Abcam, ab50887), RAD21 (Abcam, ab992), CTCF (Cell Signaling, 2899S), H3K4me1 (Active Motif, 39297), H3K4me3 (Active Motif, 39159), H3K27ac (Active Motif, 39133) and p300 (Santa Cruz).

Techniques: Recombinant, Membrane, Clinical Proteomics, Purification, Derivative Assay, Knock-Out, Protease Inhibitor, DNA Extraction, Virus, Hybridization, Library Quantification, Multiplex Assay, Immunoprecipitation, Reporter Assay, Capture-C, Sequencing, CRISPR, Expressing, Plasmid Preparation, In Situ, Software, Imaging, Microscopy

Journal: Immunity

Article Title: A PGE 2 -MEF2A axis enables context-dependent control of inflammatory gene expression

doi: 10.1016/j.immuni.2021.05.016

Figure Lengend Snippet:

Article Snippet: Protease Inhibitor Cocktail (100X) , Cell Signaling Technology , 5871.

Techniques: Control, Virus, Recombinant, CRISPR, Produced, Plasmid Preparation, DNA Purification, Staining, Saline, Protease Inhibitor, Modification, Cell Culture, SYBR Green Assay, Reverse Transcription, TA Cloning, Sequencing, Enzyme-linked Immunosorbent Assay, Luciferase, Multiplex Assay, DNA Library Preparation, Knock-In, Software

Journal: Molecular cell

Article Title: The Dystonia Gene THAP1 Controls DNA Double Strand Break Repair Choice

doi: 10.1016/j.molcel.2021.03.034

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: SsoAdvanced TM Universal SYBR Green Supermix , Bio-Rad , Cat# 1725271.

Techniques: Purification, Blocking Assay, Virus, Bacteria, Expressing, CRISPR, Knock-Out, Recombinant, Transfection, Cloning, PCR Cloning, Protease Inhibitor, Ligation, Library Quantification, Selection, Flow Cytometry, Cell Viability Assay, cDNA Synthesis, SYBR Green Assay, Cell Culture, Mutagenesis, Illumina Sequencing, Software, Microscopy, Imaging, Irradiation