Journal: The Journal of Cell Biology

Article Title: Epigenetic deprogramming by disruption of CIZ1-RNA nuclear assemblies in early-stage breast cancers

doi: 10.1083/jcb.202409123

Figure Lengend Snippet: Corrupted CIZ1–Xi assemblies in breast cancer cells. (A) Female primary human breast epithelial cells (HMECs) stained for CIZ1 via its C-terminal anchor domain (AD, green), and co-localization with H2AK119ub1 (red) as a marker of Xi chromatin. DNA is blue. Inset, example nucleus with CIZ1–AD and H2AK119ub1 shown individually in grayscale. Bar is 10 μm. (B) Frequency of cells with discrete nuclear CIZ1 assemblies, detected via CIZ1-AD (blue) or CIZ1-RD (red) in cycling populations of the indicated breast-derived cell types. Error bars show SEM. A reduced frequency of CIZ1–Xi assemblies is observed in non-cancer breast cell line MCF-10A and cancer cell line MCF7, while in the more aggressive BT-474 and MDA-MB-231 cancer cells, large CIZ1 SMACs are rare for both RD and AD epitopes, and in SK-BR-3 populations only detectable via the AD epitope. In all four of the cancer lines, the appearance of those assemblies that are detected is less compact and coherent (see part D). (C) Model showing multivalent interaction between CIZ1 N- and C-terminal RNA interaction domains, and RNAs including Xist in the vicinity of the inactive X chromosome . (D) Example immunofluorescence images of CIZ1-RD (red) and CIZ1-AD (green) in HMEC and the indicated breast cancer cell lines, after pre-fixation wash with detergent-containing buffer (Det. only), or after high-salt extraction (Det./high salt). Right, nuclei in which RD and AD are shown individually in grayscale. Bar is 10 μm. The RD and AD epitopes were differentially detected or extracted in some cases, indicating that they are not always part of the same polypeptide (for example compare nucleus-wide RD in SK-BR-3 cells, in detergent-treated cells to detergent/high-salt treated cells). (E) CIZ1 exon-specific TPMs from four breast cancer (MCF7, BT-474, SK-BR-3, and MDA-MB-231) and one normal breast-tissue derived cell model (MCF10A), normalized to the first translated exon (exon 2), showing imbalanced domain expression, favoring the C-terminal anchor domain (AD). Exon map is aligned with protein domains (see also ), and the location of epitopes used to report on CIZ1-AD (green, Ab87) or CIZ1 replication domain (RD, red, Ab1793) are shown. Below, the relative frequency of reads aligning to human CIZ1 exon 10, demonstrating consistent coverage in the normal MCF10A line, and a transition in the cancer cell lines within exon 10, at the location of an alternative transcription start site (see also ).

Article Snippet: Anti-human CIZ1 monoclonal antibody 87 was generated by Fujirebio Diagnostic Antibodies (FDAB).

Techniques: Staining, Marker, Derivative Assay, Immunofluorescence, Extraction, Expressing

Journal: The Journal of Cell Biology

Article Title: Epigenetic deprogramming by disruption of CIZ1-RNA nuclear assemblies in early-stage breast cancers

doi: 10.1083/jcb.202409123

Figure Lengend Snippet: (Related to and ). CIZ1 domains, transcript levels , and domain expression in common solid tumors. (A) Protein domain map aligning human ( NP_001124488.1 ) and mouse ( NP_082688.1 ) CIZ1. Numbers correspond to amino acids encoded at exon boundaries. The domains highlighted are: Prion-like domains 1 and 2 (PLD1 and PLD2, purple) at positions 1–78 and 360–451, respectively (human), and positions 1–67 and 361–399, respectively (mouse), 10 three zinc fingers (ZnF_C2H2 SM00355, ZF_C2H2 sd00020, and ZF_C2H2 sd00020, blue) at positions 593–617, 656–676, and 687–709, respectively (human), and 537–561, 600–620, and 631–653, respectively (mouse), an acidic domain (red) containing a concentrated area of aspartates and glutamates at position 741–761 (human) and 689–709 (mouse), and a matrin-3 homology domain (ZnF_U1 smart0045, yellow) at position 796–831 (human) and 746–770 (mouse). Box shows % identity at the amino acid level across these domains. Human and mouse CIZ1 are 65% identical at the protein level, with identity concentrated in the conserved domains (up to 96%). (B) Bright-field images of breast-derived cell types ordered based on phenotype, with corresponding hormone and growth factor receptor status. The bar is 100 μm. (C) CIZ1 locus in Homo sapiens with corresponding exon numbers. Potential CIZ1 alternative transcription start sites (TSSs) in exons 10 and 11 predicted in the FANTOM5 project are indicated (red stars). The coding sequence would be expected to begin at a methionine in exon 11. The chromatin landscape in human mammary epithelial cells (HMEC), a cervical cancer cell line (HeLa) and a breast cancer cell line (MCF7) is shown below. Diagram generated using UCSC genome browser . (D) Total CIZ1 TPM derived from the indicated number of cancer (C) and normal (N) tissues in TCGA compared using GEPIA for the indicated disease types. No significant difference is detected (where log 2 FC was >1, and P value <0.05) when comparing all amalgamated transcripts that map to the CIZ1 gene (unresolved by exon). (E) Relative expression of exons 7 (red) and 16 (blue), normalized to the average of three unmatched control samples for each of six common solid tumor types in multi-tissue cDNA array CSRT101. Individual patient data plus the average of the controls calibrated to 1 (Av.C, left) and data aggregated by disease stage (0–IV, right) are shown. 0 represents histologically normal tissue. Individual sample information for all arrays is given in .

Article Snippet: Anti-human CIZ1 monoclonal antibody 87 was generated by Fujirebio Diagnostic Antibodies (FDAB).

Techniques: Expressing, Zinc-Fingers, Derivative Assay, Sequencing, Generated, Control

Journal: The Journal of Cell Biology

Article Title: Epigenetic deprogramming by disruption of CIZ1-RNA nuclear assemblies in early-stage breast cancers

doi: 10.1083/jcb.202409123

Figure Lengend Snippet: Elevated CIZ1 anchor domain expression in primary cancers. (A) Exon structure of CIZ1 based on human reference sequence NM_012127.2 showing all 16 translated exons (2–17), and those subject to alternative splicing (pink) ( ; ; ; ; ; ). Alternative untranslated exons 1’s are not shown. The location of amplicons detected by quantitative RT-PCR detection tools (four Taqman primer/probe sets; DT5 and DT7 which detect the 5′ end of CIZ1 transcripts, and DT14 and DT16 which detect the 3′ end) are indicated. Below, the dot plot shows the comparison of outputs with the indicated pairs applied to 46 human tissue-derived cDNAs. Pearson’s correlation coefficients show strong agreement between exons 5 and 7, and between 14 and 16, but poor agreement between exons 7 and 16, or 5 and 14, indicating that the 5′ and -3′ ends of CIZ1 are typically imbalanced at the transcript level. (B) Relative quantification (RQ) of CIZ1 exon 7 (red) and CIZ1 exon 16 (blue) in primary human breast tissue-derived cDNAs in arrays BCRT103 and CSRT101 ( n = 60, all female). Box and whisker plots show results aggregated by clinical stage (0–IV), calibrated to the average of the stage 0 samples for each amplicon where 0 represents histologically normal tissue. Significance indicators show comparisons between amplicons by t test, where ns is not significant, *P < 0.05, **P < 0.01, ***P < 0.001. Individual sample values are given in . (C) As in B for human tissue-derived cDNAs in arrays MERT101 (melanoma, n = 43), HCRT103, CSRT101 (colon, n = 39), and CSRT103 (bladder, n = 24). (D) CIZ1 exon expression in TCGA breast cancer samples, separated by clinical stage and normalized to individual exon 7 expression. At all stages, 5′ and 3′ expression is significantly different, with 3′ elevation from around exon 10. Comparison of transcript levels in exon 5 to 14 or 16 (arrows) is by Mann–Whitney U test. Error bars show SEM. n = 1,087, 99% female. (E) Control analysis showing TPMs in a subset of 10 stage 2 TCGA breast cancer patients that exhibit the most marked 3′ end bias for CIZ1, mapped to CIZ1 exons, normalized to exon 7. Left, TPMs from the same patients for estrogen receptor alpha (ERα/ESR1) normalized to its exon 5, and TP53 normalized to its exon 6, showing relative exon coverage and lack of 3′ over-representation.

Article Snippet: Anti-human CIZ1 monoclonal antibody 87 was generated by Fujirebio Diagnostic Antibodies (FDAB).

Techniques: Expressing, Sequencing, Alternative Splicing, Quantitative RT-PCR, Comparison, Derivative Assay, Whisker Assay, Amplification, MANN-WHITNEY, Control

Journal: The Journal of Cell Biology

Article Title: Epigenetic deprogramming by disruption of CIZ1-RNA nuclear assemblies in early-stage breast cancers

doi: 10.1083/jcb.202409123

Figure Lengend Snippet: Dispersal of endogenous CIZ1 Xi SMACs by ectopic CIZ1 anchor domain. (A) Diagram of murine CIZ1 full-length protein showing prion-like domains (PLD) , zinc fingers (ZF), acidic domain (AcD), and Matrin 3 homology domain (MH3, see also ). Below, C-terminal protein fragments C275 and C181 correspond to the terminal 275 and 181 amino-acids respectively, both bearing an N terminal GFP tag. CIZ1 RD antibody (red) was raised against a fragment of CIZ1 outside of C275 and does not detect the transgenes. (B) Left, example images of CIZ1–Xi assemblies in D3T3 cells, showing three categories of nuclei with either large discrete CIZ1 SMACs (type 1, upper), no detectable CIZ1 SMAC (type 3, lower), or intermediate assemblies (type 2, middle), which include those that are either dispersed into multiple smaller foci or diminished in overall size or intensity. CIZ1 is red, DNA is blue. Right, frequency of cells with type 1, 2, or 3 CIZ1 Xi assemblies in untransfected (UT) populations, compared to those expressing empty GFP vector, C275 or C181. N is replicate analysis, with total nuclei inspected shown in parentheses. Error bars show SEM. For cycling cells (upper), no difference in frequencies was observed between untransfected (UT) and empty vector cells (type 1 P = 0.72, type 2 P = 0.19, type 3 P = 0.75), but dispersal was observed in C181 expressing cells compared with UT (type 1 P = 1.4 × 10 −5 , type 2 P = 0.0036, type 3 P = 0.00032). For contacted cells (lower), none or limited differences in frequencies were observed between UT and C181 expressing cells (type 1 P = 0.098, type 2 P = 0.34, type 3 P = 0.043). All comparisons of replicate analyses are by unpaired t test. (C) Box and whisker plots showing CIZ1-RD fluorescence intensity per nucleus in untransfected (UT, gray) and C181 transfected (green) WT female PEFs, showing no difference in means but a significant reduction in maxima. n is nuclei measured, comparison by T test. (D) Area occupied by Xist FISH, calculated as % of nuclear area delineated by DAPI stain, in cells expressing GFP-C181 or C275, and untransfected cells (UT) in the same populations. n is nuclei measured, comparisons by T test. (E) The intensity of nuclear Xist FISH signal, in the same cells as D, showing intensity maximas and means as box and whisker plots. Below are example images (see also ) showing Xist (red) in D3T3 cells with and without expression of GFP-C181 for 24 h. Insets illustrate compact Xist in an untransfected cell and dispersed Xist in a transfected cell (green). DNA is blue in the main image and used to create nuclear outlines (dotted lines) in insets, the bar is 5 μm. (F) Field images showing untransfected and transfected cycling and contact inhibited D3T3 cells, illustrating the effect on endogenous CIZ1 status at Xi (red). Arrows point to resistant CIZ1 Xi assemblies in contact-inhibited cells. Below are flow cytometry profiles of populations stained with propidium iodide, illustrating G1/G0 enrichment in the contacted cell population. (G) Impaired reformation of CIZ1 SMACs after release from arrest in M phase in D3T3 cells transduced with C181 compared with vector control. Left, CIZ1–Xi assembly frequency 1–5 h after release. Right, box and whisker plot showing maximum fluorescence intensity per nucleus at 4 h, where n indicates the number of nuclei measured in each group. Mann–Whitney U test, P = 6.1 × 10 −6 . (H) Illustration showing the time window of CIZ1 Xi SMAC assembly early in the G1 phase and the point of cell cycle arrest after exposure to nocodazole.

Article Snippet: Anti-human CIZ1 monoclonal antibody 87 was generated by Fujirebio Diagnostic Antibodies (FDAB).

Techniques: Zinc-Fingers, Expressing, Plasmid Preparation, Whisker Assay, Fluorescence, Transfection, Comparison, Staining, Flow Cytometry, Transduction, Control, MANN-WHITNEY

Journal: The Journal of Cell Biology

Article Title: Epigenetic deprogramming by disruption of CIZ1-RNA nuclear assemblies in early-stage breast cancers

doi: 10.1083/jcb.202409123

Figure Lengend Snippet: (Related to ). Cell cycle analysis and anchor domain mutagenesis. (A) Field views of Xist (red) in cycling D3T3 cells, 24 h after transfection with GFP-C181 or C275 (green) as indicated. DNA is blue. The bar is 10 μm. (B) CIZ1–Xi assembly frequency 1–5 h after release from cell cycle arrest in S phase (thymidine) or M phase (nocodazole). N is 2–4 as indicated, number of nuclei inspected at each time point is given ( n ). Comparisons between 1 and 5 h t test, where P = 0.45 for S phase and P = 1.15 × 10 −6 for M–G1 phase. (C) Map of C181 deletion constructs, showing excluded sequences in single letter code. These exclude the MH3 domain (conserved domain ZnF_U1 smart00451) or the fully human/mouse conserved sequence downstream of the MH3 domain (ΔNALTALF) or the murine equivalent of the eight amino acids previously implicated in lung cancer (CIZ1B) , or the terminal 37 amino acids (Δ37). We also evaluated a fragment encompassing the MH3 domain but lacking sequences up and downstream (I122) . Numbers indicate amino acid at boundaries relative to murine full-length CIZ1. AcD, acidic domain (red), and MH3, matrin 3 homology domain (yellow). (D) Example images of D3T3 cells expressing GFP-tagged C181-derived deletion mutants, without (total) and with (detergent-resistant) prefixation wash with 0.05% Triton X-100. The percentages show the proportion of transfected cells in each population with nuclear C181 or derivative, revealing the degree of sensitivity to extraction. The bar is 5 μm. (E) Effect of fragments on the frequency of endogenous CIZ1–Xi assemblies in D3T3 cells. Compared with C181, only ΔMH3 was perturbed in its ability to disperse endogenous CIZ1 (P = 0.011). All other deletion mutants retained similar DNF capability to C181 (ΔNALTALF P = 0.96, Δ37 P = 0.64, CIZ1B P = 0.99, I122 P = 1). N shows replicate analyses with total nuclei inspected in parentheses. Comparisons are by one-way ANOVA. Error bars show SEM. (F) Left, box and whisker plot showing normalized endogenous CIZ1-RD fluorescence intensity per nucleus in female WT PEFs, either untransfected (UT) or with C181, or derived deletion mutant ΔMH3, showing reduced potency of ΔMH3 compared to C181 (P = 0.002, t test). Right, mean intensity measures are ordered low to high for endogenous CIZ1 in UT, C181, and ΔMH3 expressing cells. Below are example images of cells stained for endogenous CIZ1 (red), with and without ectopic GFP-C181 or GFP-ΔMH3. Bar is 5 μm. The inset shows surviving Xi assemblies in grayscale.

Article Snippet: Anti-human CIZ1 monoclonal antibody 87 was generated by Fujirebio Diagnostic Antibodies (FDAB).

Techniques: Cell Cycle Assay, Mutagenesis, Transfection, Construct, Sequencing, Expressing, Derivative Assay, Extraction, Whisker Assay, Fluorescence, Staining

Journal: The Journal of Cell Biology

Article Title: Epigenetic deprogramming by disruption of CIZ1-RNA nuclear assemblies in early-stage breast cancers

doi: 10.1083/jcb.202409123

Figure Lengend Snippet: Effect of CIZ1 anchor domain on histone posttranslational modifications. (A) Graphs show the frequency of endogenous CIZ1–Xi assemblies in a cycling population of female D3T3 cells, comparing transfected and untransfected cells in the same population. Endogenous CIZ1 assemblies are detected via CIZ1-RD and classified into three categories; present, absent or intermediate. Middle and lower graphs show the frequency of repressive histone marks in cells that are, or are not transfected with GFP-C181. N is replicate analyses with nuclei scored in parentheses. Comparisons are by t test. For endogenous CIZ1 in UT and C181 cells P = 0.00023, for H3K27me3 P = 0.60, for H2AK119ub1 P = 0.0073. Error bars show SEM. (B) As in A, except that all data is derived from analysis of female primary embryonic fibroblasts (PEFs) at passages 2–3. For endogenous CIZ1 in UT and C181 cells P = 0.00033 for H3K27me3 P = 0.79, for H2AK119ub1 P = 0.016, performed on present (type 1) categories. Also shown is the effect of 5 μM PR619 on H2AK119ub1 loss, where P = 0.0099 for the no CIZ1 category (type 3). Error bars show SEM. (C) Example images of endogenous CIZ1 and histone marks (red) in untransfected (UT) and C181 transfected (green) WT PEF populations. The bar is 10 μm. (D) Lentivirus encoding C181 and/or ZSGreen was used to infect three independent populations of WT murine primary embryonic fibroblasts (PEFs) at passage 1–2. Below, expression was verified by western blot of ectopic CIZ1 (exon 17) and beta-actin in whole cell lysates over 3 days, compared to untreated control populations (UT) at days 1 and 3. Below right, live cell images of ZsGreen and brightfield images of PEFs at day 2 after transduction. Bar is 50 μm. (E) Comparison of vector-only populations to those transduced with C181 showing the frequency of cells with CIZ1–Xi assemblies (gray), H3K27me3 (red) or H2AK119ub1 (blue). n denotes replicate analyses with total nuclei inspected in parentheses. PEF cell populations are in gray. Comparisons are by unpaired t test where P < 0.001 in all cases. Error bars show SEM. Below are box and whisker plots showing mean nuclear intensity measures for cells transduced with C181 or vector control, normalized to the mean of vector-only control cells. Source data are available for this figure: .

Article Snippet: Anti-human CIZ1 monoclonal antibody 87 was generated by Fujirebio Diagnostic Antibodies (FDAB).

Techniques: Transfection, Derivative Assay, Expressing, Western Blot, Control, Transduction, Comparison, Plasmid Preparation, Whisker Assay

Journal: The Journal of Cell Biology

Article Title: Epigenetic deprogramming by disruption of CIZ1-RNA nuclear assemblies in early-stage breast cancers

doi: 10.1083/jcb.202409123

Figure Lengend Snippet: Effect of CIZ1 anchor domain on gene expression. (A) Possible mechanisms by which CIZ1 assemblies might influence H2AK119ub1 dynamics on Xi chromatin. Recruitment model: CIZ1– Xist assemblies contribute to recruitment or activation of PRC1, supporting H2AK119ub1 deposition. Shield model: Multiple CIZ1 dimers and RNAs coalesce to form a molecular shield at the Xi which blocks access to deubiquitinating enzymes, supporting H2AK119ub1 preservation. (B) Frequency of cells with H2AK119ub1 at the Xi in the vector-only population, and cells transduced with C181, without and with the DUB inhibitor PR619 (5 μM). N is replicate analyses with total nuclei inspected in parentheses. Comparisons are by unpaired t test. Error bars show SEM. Below are example images taken under standardized conditions showing H2AK119ub1 in red in C181-transduced WT primary embryonic fibroblasts. (C) Restoration of H2AK119ub1 enrichment at Xi in CIZ1 null primary embryonic fibroblasts by PR619. In untreated cells ∼10% of cells have H2AK119ub1 enriched Xi’s, which increased to ∼35% within 24 h of treatment, while H3K27me3 remains unchanged. Error bars show SEM. Right, example images of H2AK119ub1 in CIZ1 null primary embryonic fibroblasts. (D) Differentially expressed genes in C181 expressing PEFs, compared to vector control, showing log 2 fold change in FPKM against false-detection rate (FDR) corrected q value, and inclusion threshold of q < 0.05 and absolute log 2 FC > 1. (E) Gene set enrichment analysis of all gene sets derived from chemical or genetic perturbation of murine cells (mCGP). Significance indicator is plotted against % genes in overlap for the top 50 sets returned by C181-induced UP genes and C181-induced DN genes. Those linked with TGFβ or breast cells are highlighted in blue and orange respectively, and set identifiers are given in gray. Source data is given in . (F) Heat maps showing C181 DEGs (left, q < 0.05 log 2 FC 1), all genes in mammary stem cell set M2573 (middle), and all genes in TGFβ target set M2446 (right), where C181-induced fold change of +1.5 or over is maximally red and less than −1.5 is maximally blue. Gene names and source data are given in .

Article Snippet: Anti-human CIZ1 monoclonal antibody 87 was generated by Fujirebio Diagnostic Antibodies (FDAB).

Techniques: Gene Expression, Activation Assay, Preserving, Plasmid Preparation, Transduction, Expressing, Control, Derivative Assay

Journal: The Journal of Cell Biology

Article Title: Epigenetic deprogramming by disruption of CIZ1-RNA nuclear assemblies in early-stage breast cancers

doi: 10.1083/jcb.202409123

Figure Lengend Snippet: Gene expression in TCGA breast cancers with elevated CIZ1 anchor domain. (A) Classification of TCGA breast tumor transcriptomes based on the ratio of CIZ1 AD (exon 14) to RD (exon 5) to create a DNF index comprising groups A–D, shown after segregation by tumor stage. See also . (B) Differentially expressed genes between groups A (greater than twofold elevation of AD) and C (equal ±10%), showing 241 UP (log 2 FC ≥ 1) and 1367 DN (log 2 FC ≤ 1), where q < 0.05. Inset, pie charts show the proportion that are lncRNAs (green) or protein-coding genes (gray). (C) TCGA breast cancers are subdivided by stage, showing representation across the DNF index as % (see also ). (D) Heat map showing all differentially expressed genes derived from comparison of groups A and C, and their representation across stages I–IV. Inset, a highlight of a small subset of mostly enzyme encoding genes whose expression is suppressed in early stages but which switch to UP genes in stage IV disease. (E) Example chromosomes 1 and 9 showing, top, differentially expressed lncRNAs (green) returned by comparison of TCGA breast tumors with DNF index A (elevated AD) compared with C (balanced RD and AD). Unaffected genes are shown in gray. Middle, as above for protein-coding genes (yellow). Lower, chromatin accessibility was revealed by ATACseq in eight group A tumors compared with 15 group C tumors, with non-significant intervals in gray and differentially accessible intervals in blue. ATACseq peaks are evident across all chromosomes, and within cluster regions are exclusively UP. The cluster region is marked with a box (10 Mb).

Article Snippet: Anti-human CIZ1 monoclonal antibody 87 was generated by Fujirebio Diagnostic Antibodies (FDAB).

Techniques: Gene Expression, Derivative Assay, Comparison, Expressing

Journal: The Journal of Cell Biology

Article Title: Epigenetic deprogramming by disruption of CIZ1-RNA nuclear assemblies in early-stage breast cancers

doi: 10.1083/jcb.202409123

Figure Lengend Snippet: Affected gene clusters, lncRNA enrichment, and chromatin accessibility. (A) Chromosomal locations of differentially expressed genes derived from a comparison of TCGA CIZ1 groups A and C (UP orange, DN gray, q < 0.05). Centromere positions in black. Circled clusters are also shown in . Right, two example cluster regions on chromosomes 1 (q22), and 9 (q34). LncRNAs are green, and protein coding genes in yellow. The CIZ1 locus itself is within the circled UP cluster at 9q34. (B) Pie charts show the proportion of UP and DN genes by chromosome, highlighting the complete absence of UP genes on 18 and high representation on 19. (C) Fold gene enrichment in the indicated 10 Mb clusters compared with chromosomal average for genes (black), and those that are UP (yellow/green) or DN (gray). Protein coding (left) and lncRNAs (right) are shown separately. (D) Genome-wide ATACseq differences between group A and group C TCGA breast tumors, showing all intervals in gray and differently accessible intervals in blue (absolute log 2 FC ≥ 1, q < 0.05). Over 400 sites are significantly more exposed compared with 21 that are less exposed. (E) Illustration showing localized CIZ1–RNA assemblies surrounding and modulating access to, underlying chromatin.

Article Snippet: Anti-human CIZ1 monoclonal antibody 87 was generated by Fujirebio Diagnostic Antibodies (FDAB).

Techniques: Derivative Assay, Comparison, Genome Wide

Journal: The Journal of Cell Biology

Article Title: Epigenetic deprogramming by disruption of CIZ1-RNA nuclear assemblies in early-stage breast cancers

doi: 10.1083/jcb.202409123

Figure Lengend Snippet: Resources

Article Snippet: Anti-human CIZ1 monoclonal antibody 87 was generated by Fujirebio Diagnostic Antibodies (FDAB).

Techniques: Virus, Recombinant, Transfection, Protease Inhibitor, Mutagenesis, Plasmid Preparation, Generated, Sequencing, Software, Gene Expression, Functional Assay, Inverted Microscopy

Journal: Biomedical Reports

Article Title: Extracellular vesicles modulate endothelial nitric oxide production in patients with β‑thalassaemia/HbE

doi: 10.3892/br.2025.1957

Figure Lengend Snippet: Increased NO scavenging by splenectomised mEVs. (A) Splenectomised mEVs carrying higher α-globin protein. Western blot analysis of α-globin protein from 5x10 6 mEVs obtained from splenectomised patients with β-thalassaemia/HbE (n=3) and healthy donor (n=3). Data are presented as the mean ± S.D. Statistical analysis was performed by using independent sample T-test. Full length of western blot analysis is presented in . (B) Splenectomised mEVs scavenge higher NO. A total of 2x10 6 mEVs from splenectomised patients with β-thalassaemia/HbE (n=5) and healthy donor (n=5) were injected into the purge vessel containing 50 µM DETA NONOate and measured by a NO chemiluminescence analyser. Data are presented as the mean ± S.D. Statistical analysis was performed by using a Mann-Whitney U test. N, healthy donor; BE, splenectomised β-thalassaemia/HbE patient. NO, nitric oxide; mEVs, medium extracellular vesicles; Hb, haemoglobin; N, healthy subjects; BE, β-thalassaemia/HbE patient.

Article Snippet: The membrane was then blocked with 5% skim milk for 1 h at room temperature and incubated with a mouse anti-human α-globin chain monoclonal antibody (Santa Cruz Biotechnology, Inc.), followed by incubation with an HRP-conjugated goat anti-mouse polyclonal antibody (BD Biosciences) ( ).

Techniques: Western Blot, Injection, MANN-WHITNEY