Journal: Cancer cell

Article Title: Vitamin B6 addiction in acute myeloid leukemia

doi: 10.1016/j.ccell.2019.12.002

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Mouse: NIH 3T3 cell line , ATCC , Cat# CRL-1658, RRID:CVCL_0594.

Techniques: Virus, Plasmid Preparation, Retroviral, Recombinant, Mutagenesis, RNA Extraction, Gel Extraction, Cloning, Purification, Flow Cytometry, Sequencing, CRISPR, Functional Assay, shRNA, 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: bioRxiv

Article Title: HTRA3 protease-chaperone stabilizes cathepsin B for mitochondrial POLG1 depletion in human cell ageing

doi: 10.1101/2025.07.21.647696

Figure Lengend Snippet: (A) WB of CSB, HTRA3 (left panel), and CTSB, and p21 (right panel) in BJ fibroblasts untreated and 10 days post-irradiation using ß-tubulin and GAPDH, respectively, as loading control. (B) Scheme showing the two hypotheses for the mechanism leading to senescence-related HTRA3 and CTSB overexpression. (C) Immunoblots of p21, CSB, HTRA3, CTSB, and POLG1 in BJ-5ta hTERT WT fibroblasts and 2 CRISPR-edited CSB knocked-out clones (BJ-5ta csb-/- hTERT #1 and BJ-5ta csb-/- hTERT #2). Frames show samples on the same blot; each displaying the respective GAPDH used as loading control. (D) Scheme indicating normally blocked senescence in BJ-5ta hTERT cells and activation of senescence upon irradiation at 10 Gy. WB analysis of (E) CSB, CTSB, p21, and (F) HTRA3 three days post-irradiation in BJ-5ta hTERT WT fibroblasts and BJ-5ta hTERT CSB knocked-out fibroblasts (β-tubulin was used as loading control). RT-qPCR of ( G ) p21 Waf1 and ( H ) HTRA3 and ( I ) CTSB in the same experiment and cells schematized in panel D. Samples on the same blot are framed; each frame displays the respective GAPDH or β-tubulin used as loading control. RT-qPCR: n=3 independent experiments; mean ± SD. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001; based on unpaired t test comparisons to respective non-irradiated control.

Article Snippet: Normal female human foetal lung IMR-90 fibroblasts (ATCC; CCL-186) and BJ skin fibroblasts (ATCC; CRL-2522) were cultured in minimum essential medium (MEM, Gibco) supplemented with 2 mM L-glutamin (GlutMAX), 10% FBS, 1% Penicillin–streptomycin, 1% nonessential amino acids (Gibco) and 1% sodium pyruvate (Gibco) BJ-5ta hTERT immortalized human fibroblasts (ATCC; CCL-186) cultured in a 4:1 mixture of DMEM and Medium 199 (Gibco) supplemented with 10% FBS and1% Penicillin–streptomycin.

Techniques: Irradiation, Control, Over Expression, Western Blot, CRISPR, Clone Assay, Activation Assay, Quantitative RT-PCR

Journal: AJNR: American Journal of Neuroradiology

Article Title: Efficient Transmicrocatheter Delivery of Functional Fibroblasts with a Bioengineered Collagen Gel-Platinum Microcoil Complex: Toward the Development of Endovascular Cell Therapy for Cerebral Aneurysms

doi: 10.3174/ajnr.A0593

Figure Lengend Snippet: Dependency of collagen gel contraction on fibroblast attenuation and time. Collagen gel diameter measured at time point (Dm) plotted as percentage of the initial gel diameter (Di) over a 7-day period for cell carrier devices of varying initial fibroblast (cell) attenuation. Initial gel collagen concentration was 2 mg/mL for all cell carrier devices and control devices containing no cells. Each data point is an average of triplicate samples (except for cell attenuation of 2 × 107 cells/mL) (n = 2); error bars are SDs for each data series. Day 7: 107 cells/mL construct versus 106 cells/mL construct (P < .08); 107 cells/mL construct versus 105 cells/mL construct (P < .003); 107 cells/mL construct versus 2 × 107 cells/mL construct (P > .25) (printed with permission from the Mayfield Clinic).

Article Snippet: Cell Culture Neonatal human dermal fibroblasts (Clonetics-Biowhittaker, Walkersville, Md) were subcultured, preserved in liquid nitrogen, and propagated for 5 to 10 passages before harvesting.

Techniques: Concentration Assay, Control, Construct

Journal: bioRxiv

Article Title: Distinct roles of BRCA2 in replication fork protection in response to hydroxyurea and DNA interstrand crosslinks

doi: 10.1101/811968

Figure Lengend Snippet: (A) Family pedigree showing a sibling pair with Fanconi anemia (red circles) who are compound heterozygous for BRCA2 variants c.2330dupA (maternal inheritance) and c.8524C>T (paternal inheritance). Family history of breast cancer (purple, all diagnosed in 60s and 70s), skin cancer (grey), and colon cancer (green, diagnosed at 40 years old). (B) Schematic of BRCA2 domain structure and key interacting proteins (C) Alignment of exon 20 BRCA2 DBD peptide sequence demonstrating it is evolutionary conserved across many species. In green are the aa residues modified by the patient variants, p.W2830_K2833del (c.8488-1G>A) and p.R2842C (c.8524C>T). Purple arrows indicate AA residues that contact DNA . (D) Immunoblot showing BRCA2 levels in WT (RA2985) control, FA-D1 (RA2525), and patient RA3105 and RA3106 LCLs. (E) Quantification of chromosome breaks following DEB treatment of WT (RA2985), FA-A (RA2939), and patient RA3105 and RA3106 LCLs. (F-G) Cell survival assays of patient derived lymphoblast cell lines (LCLs) RA3105, FA-A (RA2939), WT (RA2985), and FA-D1 (RA2525) after MMC and PARP inhibitor olaparib (PARPi) treatment. Relative cell survival was normalized to untreated controls to give percent survival. Error bars indicate s.d. (H) Quantification of chromosome breaks following MMC treatment of BJ wild type fibroblasts, FA-A patient fibroblasts, and HSC62 fibroblasts. (I) Cell survival of HSC62 (c.8488-1G>A) fibroblasts compared to BJ WT fibroblast, FA-A patient fibroblast, complemented FA-A patient cells (RA3087) expressing wild type FANCA (FA-A+A) or empty vector (FA-A+EV). Cells were treated with increasing concentrations of MMC. Relative cell survival was normalized to untreated controls to give the percent survival. Error bars indicate s.d. (J) Cell survival of MMC treated HSC62 uncorrected patient cell line (HSC62 mut ) compared to BJ WT fibroblast and CRISPR/Cas9 corrected wild type HSC62 (HSC62 WT ) clones 1-3. (K-L) Cell survival of BJ WT fibroblasts, and CRISPR/Cas9 targeted BJ fibroblasts: BJ WT fibroblast clone (BRCA2 WT ), c.8488-1G>A BJ clones (BRCA2 8488-1G>A ), c.8524C>T BJ clones (BRCA2 8524C>T) , and exon 20 BRCA2 frameshift mutant (BRCA2 Trun. ). Cells were treated with increasing concentrations of MMC or PARPi. Error bars indicate s.d.

Article Snippet: Patient-derived fibroblast cell lines ( ) and BJ foreskin normal control fibroblasts (ATCC) were transformed by expression of HPV16 E6E7 and immortalized with the catalytic subunit of human telomerase (hTERT).

Techniques: Sequencing, Modification, Western Blot, Control, Derivative Assay, Expressing, Plasmid Preparation, CRISPR, Clone Assay, Mutagenesis

Journal: bioRxiv

Article Title: Distinct roles of BRCA2 in replication fork protection in response to hydroxyurea and DNA interstrand crosslinks

doi: 10.1101/811968

Figure Lengend Snippet: (A-B) Chromatograms of Sanger sequencing of DNA derived from a sibling pair and parents confirming the presence of BRCA2 c.2330dupA and c.8524C>T variants identified by whole exome sequencing (WES). (C) BRCA2 structure of the DBD illustrating the location of p.W2830_K2833del and p.R2842C patient variants at the base of the Tower domain and OB2. Structure adapted from Yang et al ., 2005. (D) Immunoblot analysis for FANCI ubiqutination following treatment with 1μM MMC for 24h of. WT (RA2985), FA-A (RA2939), and patient RA3105 and RA3106 LCLs. (E) Metaphase for RA2985 and RA3105 following DEB treatment. (F-G) Cell survival assays of patient derived lymphoblast cell line (LCLs) RA3105, FA-A (RA2939), WT (RA2985), and FA-D1 (RA2525) after (DEB) and camptothecin (CPT) treatment. Survival assays were performed in triplicate. Cells were treated with increasing concentrations of genotoxic agents and counted after 7-10 days in culture. Relative cell survival was normalized to untreated controls to give percent survival. Error bars indicate s.d. (H-N) Cell survival of HSC62 fibroblasts (c.8488-1G>A) to indicated agent compared to BJ WT fibroblast, FA-A patient fibroblast (RA3087), FA-A complemented patient cells expressing wild type FANCA (FA-A+A) or empty vector (FA-A+EV), RAD50 patient fibroblast or FA-D2 (BRCA2/FANCD1) patient fibroblast (FA-D1). Cell survival assays were performed in triplicate. Cells were treated with increasing concentrations of indicated agent. Cell survival was determined by counting cells after 7-9 days in culture. Relative cell survival was normalized to untreated controls to give the percent survival. Error bars indicate s.d. (O) Quantification of chromosome breaks following DEB treatment of BJ wild type fibroblasts, FA-A (FANCA) patient fibroblasts (FA-A mut ), and HSC62 fibroblasts. (P) Chromatograms of PCR amplified gDNA of CRISPR/Cas9 targeted HSC62 fibroblasts. Gene editing reverted the c.8488-1G>A variant either to homozygous WT (HSC62 WT ) or heterozygous WT (HSC62 mut/WT ) at the endogenous locus in HSC62 patient cells. The silent variant that was incorporated to destroy the CRISPR PAM sequence is indicated. (Q)cDNA analysis of HSC62 clones with either homozygous or heterozygous correction of the c.8488-1G>A variant demonstrating rescue of the 12bp deletion of exon 20 that results from alternate splicing. (R) Immunoblot showing BRCA2 levels in CRISPR/CAS9 corrected patient cell line HSC62 WT , uncorrected HSC62 cells (HSC62 mut ), and RA2630 FA-R (RAD51/FANCR) patient fibroblasts. (S-T) Cell survival of HSC62 uncorrected patient cell line (HSC62 mut ) compared to BJ WT fibroblast and CRISPR/Cas9 corrected wild type HSC62 (HSC62 WT ) clone. Error bars indicate s.d.

Article Snippet: Patient-derived fibroblast cell lines ( ) and BJ foreskin normal control fibroblasts (ATCC) were transformed by expression of HPV16 E6E7 and immortalized with the catalytic subunit of human telomerase (hTERT).

Techniques: Sequencing, Derivative Assay, Western Blot, Expressing, Plasmid Preparation, Amplification, CRISPR, Variant Assay, Clone Assay

Journal: bioRxiv

Article Title: Distinct roles of BRCA2 in replication fork protection in response to hydroxyurea and DNA interstrand crosslinks

doi: 10.1101/811968

Figure Lengend Snippet: (A) cDNA sequencing of BRCA2 DBD CRISPR/Cas9 targeted BJ fibroblasts demonstrating that homozygous c.8488-1G>A variant in BJ fibroblast clones result in the usage of an alternative splice site donor and a 12bp deletion at the start of BRCA2 exon 20 (as seen in patient derived HSC62 fibroblasts). (B) cDNA sequencing of BRCA2 DBD CRISPR/Cas9 targeted BJ fibroblasts demonstrating the c.8524C>T missense variant and silent mutation introduced by CRISPR/Cas9 targeting. (C) Immunoblot showing BRCA2 levels in bulk BJ WT fibroblasts, BRCA2 WT fibroblast clone, c.8488-1G>A BJ clones 1-3, c.8524C>T BJ clones 1-2, and BRCA2 Trun. clones 1-2. (D-E) Cell survival of BJ WT fibroblasts, BJ WT fibroblast clone (BRCA2 WT ), c.8488-1G>A BJ clones, c.8524C>T BJ clones, and exon 20 BRCA2 frameshift mutant (BRCA2 Trun. ). Cell survival assays were performed in triplicate. Cells were treated with increasing concentrations of CPT or aphidicolin. Cell survival was determined by counting cells after 7-9 days in culture. Relative cell survival was normalized to untreated controls to give the percent survival. Error bars indicate s.d. (F-G) Representative images of RAD51 foci in HSC62 uncorrected patient cell line (HSC62 mut ) compared to BJ WT fibroblast and CRISPR/Cas9 corrected wild type HSC62 (HSC62 mut/WT or HSC62 WT ) clones, 8h post IR and 24h post MMC. Detected by immunofluorescence with anti-RAD51antibody. Quantification in and . (H) Quantification of RAD51 foci in isogenic BJ fibroblasts clones 8h, 24h and 48h following 1h treatment with 3 μM MMC of BJ WT fibroblasts, BJ WT fibroblast clone (BRCA2 WT ), c.8488-1G>A BJ clones 2-3, c.8524C>T BJ clones 1-2, and a BRCA2 truncation mutant, c.8531dupA ( BRCA2 Trun ). Error bars indicate s.d. of three independent experiments (≥200 cells per experiment). (I) Representative images of RAD51 foci in isogenic BJ fibroblasts clones, 24h post 1h treatment with 3 μM MMC, detected by immunofluorescence with anti-RAD51antibody. Third row images are individual cells enlarged to better demonstrate differences in RAD51 foci size. Quantified in .

Article Snippet: Patient-derived fibroblast cell lines ( ) and BJ foreskin normal control fibroblasts (ATCC) were transformed by expression of HPV16 E6E7 and immortalized with the catalytic subunit of human telomerase (hTERT).

Techniques: Sequencing, CRISPR, Variant Assay, Clone Assay, Derivative Assay, Mutagenesis, Western Blot, Immunofluorescence

Journal: bioRxiv

Article Title: Distinct roles of BRCA2 in replication fork protection in response to hydroxyurea and DNA interstrand crosslinks

doi: 10.1101/811968

Figure Lengend Snippet: (A) Immunofluorescence images of RAD51 foci, 8h following 12 Gy ionizing radiation (IR) of BJ WT fibroblast and patient derived HSC62 fibroblast, detected with anti-RAD51 antibody. Third row images are individual cells enlarged to better demonstrate differences in RAD51 foci size. (B) Quantification of RAD51 foci 1h, 8h, and 24h following 12 Gy IR of BJ WT fibroblast and HSC62 fibroblast. Error bars indicate s.d. of two independent experiments (≥200 cells per experiment). (C) Quantification of RAD51 foci 8h after 12 Gy IR of BJ WT fibroblast, wild type HSC62 - (HSC62 WT ) clones 1-3, and HSC62 uncorrected patient cell line (HSC62 mut ). (D) Quantification of RAD51 foci 24h following 1h treatment with 3 µM MMC. Error bars indicate s.d. of three independent experiments (≥200 cells per experiment). (E) Quantification of RAD51 foci in isogenic BJ fibroblasts clones at 1h, 8h and 24h following 6 Gy IR of BJ WT fibroblasts, BJ WT fibroblast clone (BRCA2 WT ), BRCA2 8488-1G>A BJ clones 2-3, BRCA2 8524C>T BJ clones 1-2, and a BRCA2 homozygous truncation mutant, c.8531dupA (BRCA2 Trun ). Error bars indicate s.d. of three independent experiments (≥200 cells per experiment) (F) Representative images of RAD51 foci in isogenic BJ fibroblasts clones, 8h post 6 Gy IR, detected by immunofluorescence with anti-RAD51 antibody. Third row images are individual cells enlarged to better demonstrate differences in RAD51 foci size. (G) Quantification of RPA foci 24h following 1h treatment with 3 μM MMC of BJ WT fibroblast, CRISPR/Cas9 corrected wild type HSC62 clones (HSC62 WT ), and HSC62 uncorrected patient cell line (HSC62 mut ). (H) Quantification of RPA foci 24h following 1h treatment with 3 μM MMC in HSC62 mut cells depleted of DNA2, MRE11, EXO1, CTIP, WRN, or BLM by siRNA compared to luciferase control (Luc). Error bars indicate s.d. of four independent experiments. (I) Immunoblot analysis of RPA phosphorylation in isogenic BJ fibroblasts clones 24h post 1h treatment with 3 μM MMC. BRCA2 WT , BRCA2 8524C>T , and BRCA2 8488-1G>A BJ fibroblast cells were transfected with siRNA control luciferase (Luc) or siRNAs targeting DNA2 or WRN. (J-K) MMC cell survival of BJ BRCA2 WT , BRCA2 8488-1G>A , and BRCA2 8524C>T fibroblasts overexpressing (OE) WT RAD51 or empty vector (EV) control. Relative cell survival was normalized to untreated controls to give percent survival. Error bars indicate s.d.

Article Snippet: Patient-derived fibroblast cell lines ( ) and BJ foreskin normal control fibroblasts (ATCC) were transformed by expression of HPV16 E6E7 and immortalized with the catalytic subunit of human telomerase (hTERT).

Techniques: Immunofluorescence, Derivative Assay, Clone Assay, Mutagenesis, CRISPR, Luciferase, Control, Western Blot, Phospho-proteomics, Transfection, Plasmid Preparation

Journal: bioRxiv

Article Title: Distinct roles of BRCA2 in replication fork protection in response to hydroxyurea and DNA interstrand crosslinks

doi: 10.1101/811968

Figure Lengend Snippet: (A) Images of RPA foci in HSC62 uncorrected patient cell line (HSC62 mut ) compared to BJ WT fibroblast and CRISPR/Cas9 corrected wild type HSC62 (HSC62 mut/WT or HSC62 WT ) clones, 24h post 1h treatment with 3 μM MMC. Detected by immunofluorescence with anti-RPA32 antibody. Quantification in . (B)Immunoblot analysis of MRE11 and CTIP siRNA depletion for cells utilized in . (C) qRT-PCR of DNA2, EXO1, WRN, and BLM expression levels of cells in . Error bars are s.d. (D) Quantification of RPA foci in isogenic BJ fibroblasts clones 8h, 24h and 48h following 1h treatment with 3 μM MMC of BJ WT fibroblasts, BJ WT fibroblast (BRCA2 WT ), c.8488-1G>A BJ clones 2-3, c.8524C>T BJ clones 1-2, and a BJ BRCA2 truncation mutant (BRCA2 Trun. ). Error bars indicate s.d. of three independent experiments (≥200 cells per experiment). (E) Representative images of RPA foci in isogenic BJ fibroblasts clones, 24h post 1h treatment with 3 uM MMC, detected by immunofluorescence with anti-RPA32 antibody. Third row images are individual cells enlarged to better demonstrate RPA foci. Quantified in . (F) Quantification of RPA foci in isogenic BJ fibroblasts clones 24h following 1h treatment with 3 μM MMC in cells depleted of DNA2 or WRN by siRNA. Error bars indicate s.d of two independent experiments. (G-H) qRT-PCR of DNA2 and WRN expression levels of cells utilized in and . Error bars indicate s.d. (I) Immunoblot analysis of WT RAD51 overexpression in BRCA2 8524C>T and BRCA2 8488-1G>A BJ fibroblast cells used in and . (J) Quantification of RPA foci 24h following 1h treatment with 3 μM MMC in cells expressing WT RAD51 or EV control. Representative data of 3 independent experiments. (K) Quantification of RPA foci in isogenic BJ fibroblasts clones 24h following 1h treatment with 3 uM MMC in cells depleted of SLX4 or MUS81 by siRNA. Error bars indicate s.d of two independent experiments. (L) Immunoblot analysis of MUS81 depletion for cells utilized in . (M) qRT-PCR of SLX4 expression levels of cells utilized in . Error bars indicate s.d. (N-O) qRT-PCR of DNA2 and WRN expression levels of FA-A fibroblast cell lines utilized in . Error bars indicate s.d. (P-Q) qRT-PCR of DNA2 and BLM expression levels of FA-A fibroblast cell lines utilized in . Error bars indicate s.d.

Article Snippet: Patient-derived fibroblast cell lines ( ) and BJ foreskin normal control fibroblasts (ATCC) were transformed by expression of HPV16 E6E7 and immortalized with the catalytic subunit of human telomerase (hTERT).

Techniques: CRISPR, Clone Assay, Immunofluorescence, Western Blot, Quantitative RT-PCR, Expressing, Mutagenesis, Over Expression, Control

Journal: bioRxiv

Article Title: Distinct roles of BRCA2 in replication fork protection in response to hydroxyurea and DNA interstrand crosslinks

doi: 10.1101/811968

Figure Lengend Snippet: (A) Quantification of RPA foci 8h, 24h and 48h following 1h treatment with 3 μM MMC of FA patient derived fibroblasts compared to BJ wild type fibroblasts. Patient cells lines from FA complementation group FA-R (RAD51/FANCR) (FA-R mut ), FA-A (FANCA) (FA-A mut ), FA-L (FANCL) (FA-L mut ), FA-D2 (FANCD2) (FA-D2 mut ), FA-I (FANCI) (FA-I mut ), FA-J (FANCJ) (FA-J mut ), and FA-P (SLX4/FANCP) (FA-P mut ). FA-A patient complemented cell lines were generated by transducing WT FANCA cDNA or EV. Error bars indicate s.d. of two independent experiments. (B) FA-A patient cells expressing WT FANCA (FA-A+A) or empty vector (FA-A+EV) were transfected with siRNA control luciferase (Luc) or siRNAs targeting DNA2 and WRN. Quantification of RPA foci 24h following 1h treatment with 3 μM MMC. Error bars indicate s.d. of two independent experiments. (C) FA-A+EV were transfected with siRNA Luc or siRNAs targeting DNA2 and BLM. Quantification of RPA foci 24h following 1h treatment with 3 μM MMC. Error bars indicate s.d. of two independent experiments.

Article Snippet: Patient-derived fibroblast cell lines ( ) and BJ foreskin normal control fibroblasts (ATCC) were transformed by expression of HPV16 E6E7 and immortalized with the catalytic subunit of human telomerase (hTERT).

Techniques: Derivative Assay, Generated, Expressing, Plasmid Preparation, Transfection, Control, Luciferase

Journal: bioRxiv

Article Title: Distinct roles of BRCA2 in replication fork protection in response to hydroxyurea and DNA interstrand crosslinks

doi: 10.1101/811968

Figure Lengend Snippet: (A) Chromatograms of BRCA2 CRISPR/Cas9 generated HEK293T clones aligned to WT. A frameshift Exon 20 BRCA2 mutant (BRCA2 Trun. ) was generated by homozygous single base pair insertion as a result of CRISPR/Cas9 targeting. BRCA2 exon 20 variants, c.8524C>T and c.8488-1G>A, and Exon 27 p.S3291A (c.9871T>G) clones were generated by targeting the respective BRCA2 exon with CRISPR/Cas9 and a 100bp ssDNA template donor. Where applicable, silent mutations are indicated. Cell lines utilized in . (B) Immunoblot showing BRCA2 levels in WT HEK293T cells and BRCA2 mutant HEK293T clones: c.8531dupA ( BRCA2 Trun ), c.8524C>T (clones 1-2), c.8488-1G>A (clones 1-3), and p.S3291A (clones 1-3). Cells utilized in . (C) qRT-PCR of BRCA2 expression levels of cells utilized in . Error bars are s.d. (D) Immunoblot of RAD51 knockdown for HEK293T cells used in . (E) SCE assay in BJ WT fibroblast and HSC62 fibroblast following depletion of BLM. (F) Representative images of SCEs in BJ WT fibroblast and HSC62 fibroblast metaphases. (G) qRT-PCR of BLM expression levels in cells described in E. Error bars indicate s.d. (H) Sister chromatid exchange (SCE) assay in BJ BRCA2 WT and BRCA2 Trun fibroblasts following treatment with MMC (0.05 μg/ml or 0.1 μg/ml). (I) Immunoblot analysis of MRE11 depletion for cells utilized in ,F. (J) qRT-PCR of DNA2 expression levels of cells utilized in ,F. Error bars indicate s.d.

Article Snippet: Patient-derived fibroblast cell lines ( ) and BJ foreskin normal control fibroblasts (ATCC) were transformed by expression of HPV16 E6E7 and immortalized with the catalytic subunit of human telomerase (hTERT).

Techniques: CRISPR, Generated, Clone Assay, Mutagenesis, Western Blot, Quantitative RT-PCR, Expressing, Knockdown

Journal: bioRxiv

Article Title: Distinct roles of BRCA2 in replication fork protection in response to hydroxyurea and DNA interstrand crosslinks

doi: 10.1101/811968

Figure Lengend Snippet: (A) Levels of mClover positive cells were normalized to WT HEK293T (siLuc). Error bars indicate s.d. of three independent experiments performed in triplicate. (B) Sister chromatid exchange (SCE) assay in BJ WT fibroblast and HSC62 patient derived fibroblast following treatment with MMC (0.1 μg/ml or 0.2 μg/ml). (C) Isogenic BJ fibroblast BRCA2 mutants, BRCA2 Trun. , BRCA2 8524C>T and BRCA2 8488-1G>A , were analyzed for replication fork resection. Cells were labeled with DNA analogs, IdU for 20 minutes and then CldU for 20 minutes. Cells were then incubated in 6 mM HU with and without MRE11 inhibitor mirin (50 uM) for 4h before being harvested. DNA fibers were prepared and visualized by immunofluorescence detection of IdU and CldU and measured. Error bars indicate s.d. (D) Isogenic BJ fibroblast BRCA2 mutants, BRCA2 Trun. , BRCA2 8524C>T , BRCA2 8488-1G>A , and BRCA2 S3291A , were transfected with siRNA control luciferase (Luc) or siRNAs targeting DNA2 or MRE11. Cells were treated and labeled with DNA analogs as above. Error bars indicate s.d. (E) BJ fibroblast with BRCA2 variants, BRCA2 8524C>T and BRCA2 8488-1G>A , were analyzed for replication fork resection when depleted of RADX by shRNA or transduced with shRNA control (shCONT.). Cells were treated and labeled with DNA analogs as above. Data of two replicated plotted. Error bars indicate s.d. (F) Quantification of chromosome breaks in isogenic BJ fibroblast BRCA2 mutants following 5h of 6 mM HU and released into colcemid. Breakage was not significantly increased in BRCA2 8524C>T and BRCA2 8488-1G>A compared to BRCA2 WT .

Article Snippet: Patient-derived fibroblast cell lines ( ) and BJ foreskin normal control fibroblasts (ATCC) were transformed by expression of HPV16 E6E7 and immortalized with the catalytic subunit of human telomerase (hTERT).

Techniques: Derivative Assay, Labeling, Incubation, Immunofluorescence, Transfection, Control, Luciferase, shRNA, Transduction

Journal: bioRxiv

Article Title: Distinct roles of BRCA2 in replication fork protection in response to hydroxyurea and DNA interstrand crosslinks

doi: 10.1101/811968

Figure Lengend Snippet: (A) BJ fibroblast mutants BRCA2 8524C>T and BRCA2 8488-1G>A , were analyzed for replication fork resection when depleted of either SMARCAL1 or ZRANB3 by shRNA or transduced with control shRNA (shLuc). Cells were labeled with DNA analogs, IdU for 20 minutes and then CldU for 20 minutes. Cells were then incubated in 6 mM HU for 4h before being harvested. DNA fibers were prepared and visualized by immunofluorescence detection of IdU and CldU and measured. Error bars indicate s.d. (B) Quantification of RPA foci in isogenic BJ fibroblasts clones 24h following 1h treatment with 3 µM MMC in cells depleted of SMARCAL1 or ZRANB3. Error bars indicate s.d of two independent experiments. (C) Quantification of RPA foci in BJ fibroblasts clones 24h following 1h treatment with 3 uM MMC in cells depleted of HLTF. Error bars indicate s.d of two independent experiments. (D) MMC cell survival of isogenic BJ BRCA2 WT fibroblasts depleted of SMARCAL1 or ZRANB3 by shRNA or transduced with shRNA luciferase control (shLuc). Relative cell survival was normalized to untreated controls to give percent survival. Error bars indicate s.d. (E-F) MMC and CPT cell survival assay of isogenic BJ BRCA2 8488-1G>A or BRCA2 8524C>T clones depleted of either SMARCAL1 or ZRANB3 by shRNA or transduced with shRNA luciferase control (shLuc). Relative cell survival was normalized to untreated controls to give percent survival. Error bars indicate s.d.

Article Snippet: Patient-derived fibroblast cell lines ( ) and BJ foreskin normal control fibroblasts (ATCC) were transformed by expression of HPV16 E6E7 and immortalized with the catalytic subunit of human telomerase (hTERT).

Techniques: shRNA, Transduction, Control, Labeling, Incubation, Immunofluorescence, Clone Assay, Luciferase, Clonogenic Cell Survival Assay

Journal: bioRxiv

Article Title: Distinct roles of BRCA2 in replication fork protection in response to hydroxyurea and DNA interstrand crosslinks

doi: 10.1101/811968

Figure Lengend Snippet: (A) Immunoblot analysis of RADX depletion by siRNA for cells utilized in . (B) Immunoblot analysis of RADX depletion by two shRNAs for cells utilized in . (C-D) Comparison of levels of mClover positive cells of HEK293T BRCA2 mutants transfected with siRNAs or shRNAs targeting RADX or control (Luc or CONT). (E) Quantification of chromosome breaks in isogenic BJ BRCA2 WT and BRCA2 Trun fibroblasts following 5h of 6 mM HU and released into colcemid. (F) Quantification of chromosome breaks in isogenic BJ BRCA2 WT and BRCA2 S3291A following 5h of 6 mM HU and released into colcemid. (G) Immunoblot analysis of RPA phosphorylation 24h post 1h treatment with 3 uM MMC. BRCA2 8524C>T and BRCA2 8488-1G>A BJ fibroblast cells were depleted of either SMARCAL1 or ZRANB3 by shRNA or transduced with shRNA control (Luc). (H-I) Immunoblot analysis of shRNA depletion of SMARCAL1 and ZRANB3 for cells utilized in and .

Article Snippet: Patient-derived fibroblast cell lines ( ) and BJ foreskin normal control fibroblasts (ATCC) were transformed by expression of HPV16 E6E7 and immortalized with the catalytic subunit of human telomerase (hTERT).

Techniques: Western Blot, Comparison, Transfection, Control, Phospho-proteomics, shRNA, Transduction

Journal: Nature Communications

Article Title: PRC2-AgeIndex as a universal biomarker of aging and rejuvenation

doi: 10.1038/s41467-024-50098-2

Figure Lengend Snippet: a Average DNAm levels at CpGs across the whole genome (left panel), at LMRs ranked by the level of their PRC2 binding in hESCs (center panel) and within high-PRC2 regions (right panel) from 8 different tumor and normal samples. Data were obtained from TCGA WGBS datasets, (see Supplementary Data ). Error bars on the right show 95% confidence intervals for average DNAm across high-PRC2 LMRs for each sample. b Average DNAm levels at CpGs across the whole genome (left panel) and at LMRs ranked by the level of their PRC2 binding in hESCs (right panel) from young vs old human oligodendrocytes. (WGBS dataset, GSE107729, n = 6). c Average DNAm levels at CpGs across the whole genome (left panel) and at LMRs ranked by the level of their PRC2 binding in hESCs (middle panel), correlation between average DNAm at high-PRC2 LMRs and the number of passages (right panel, shaded area of line plot represents 95% CI) in in vitro cultured fibroblasts (GSE79798, n = 5). High-PRC2 LMRs are highlighted with dotted red boxes.

Article Snippet: Neonatal fibroblasts were purchased from Cell Applications (#106-05n) and old fibroblasts were isolated from skin tissue collected from deceased healthy individuals.

Techniques: Binding Assay, In Vitro, Cell Culture

Journal: Nature Communications

Article Title: PRC2-AgeIndex as a universal biomarker of aging and rejuvenation

doi: 10.1038/s41467-024-50098-2

Figure Lengend Snippet: a Average DNAm levels of neonatal and old passaged fibroblasts, and CD4 T-cells, at CpGs across the whole genome (left panel) and at the LMRs ranked by the level of EZH2-binding data of the same respective tissue (middle panels). Bottom panels show heatmaps of LMR rank number, ordered by EZH2 binding in their respective tissue (top heatmap) and in hESCs (bottom heatmap). Heatmaps are colored by EZH2 binding in the same respective tissue, i.e. purple/orange represents high and low-ranked neonatal/old LMRs ordered by neonatal/old fibroblasts PRC2 binding, respectively, and red/blue represent high and low-ranked CD4 T-cells LMRs ordered by CD4 T-cells PRC2 binding, respectively. Right panel shows the correlation of mean methylation of high-PRC2 LMRs against age or passage number. b Average methylation levels at LMRs ranked by PRC2 binding in hESCs for neonatal fibroblasts (left panels) and old fibroblasts (right panels). c Average DNAm levels of LMRs calculated from neonatal and old samples merged (all passages), ranked by PRC2 binding in hESCs (left panel) and correlation between methylation and age for high-PRC2 LMRs of both neonatal and old in vitro passaged fibroblasts (right panel). d Heatmap of normalized read density of the high-PRC2 neonatal/old LMRs in neonatal and old fibroblasts (passage 2). WGBS Neonatal and old fibroblasts were generated by our lab (GSE253987, n = 3 for neonatal samples (one donor) and n = 3 for old samples (one donor)), CD4 T-cell samples are the same as used in Fig. (6 samples 18–86 years old, GSE79798 and 2 samples 0 and 103 years old, GSE31263). ChIP data for fibroblast and CD4 T-cells were generated by our lab (GSE253987, two donors pooled at passage 2 for neonatal fibroblasts, three donors pooled at passage 2 for old fibroblasts, three donors pooled for CD4 T-cells). High-PRC2 LMRs are highlighted with dotted red boxes. Line plots with >3 samples plotted have a shaded area representing 95% CI.

Article Snippet: Neonatal fibroblasts were purchased from Cell Applications (#106-05n) and old fibroblasts were isolated from skin tissue collected from deceased healthy individuals.

Techniques: Binding Assay, Methylation, In Vitro, Generated