Journal: Nature Communications

Article Title: Loss of heterozygosity of essential genes represents a widespread class of potential cancer vulnerabilities

doi: 10.1038/s41467-020-16399-y

Figure Lengend Snippet: a Schematic of EXOSC8 SNP rs117135638 locus showing target sites for positive control, non-allele specific (NA) sgRNA and experimental, allele-specific (AS) sgRNA. Alleles appear in bold. b Crystal structure of EXOSC8 gene product, Rrp43 (gray) shows the amino acid encoded by rs117135638 (teal) lies on the surface of the Rrp43 protein near the interface with exosome complex subunit Mtr3 (orange). c Disruption of EXOSC8 in patient-derived EXOSC8 resistant (EXOSC8 R ) or EXOSC8 sensitive (EXOSC8 S ) cells expressing EXOSC8 non-allele specific (NA) positive control sgRNA or allele-specific (AS) experimental sgRNA. Unaltered alleles (black), alleles with in-frame insertions or deletions (gray), and alleles with frameshift alterations (yellow) were assessed by deep sequencing of EXOSC8 four days post-infection with sgRNA. d Immunoblot of EXOSC8 protein levels in indicated patient-derived and isogenic cell lines expressing LacZ, EXOSC8 NA, or EXOSC8 AS sgRNA ( n = 2 technical replicates of 1 biological sample). e Representative growth curves of indicated patient-derived and isogenic cell lines expressing LacZ (black), EXOSC8 NA (red), or EXOSC8 AS (blue) sgRNA, as measured by CellTiter-Glo luminescence, relative to day of assay plating. n = 5 technical replicates. Data are presented as mean values ± s.d. See Supplementary Fig. for additional biological replicates. f Immunoblot of EXOSC8 protein levels in indicated isogenic cell lines expressing LacZ, EXOSC8 NA, or EXOSC8 AS sgRNA ( n = 2 technical replicates of 1 biological sample). g Representative growth curves of indicated isogenic cell lines expressing LacZ (black), EXOSC8 NA (red), or EXOSC8 AS (blue) sgRNA, as measured by CellTiter-Glo luminescence, relative to day of assay plating. n = 5 technical replicates. Data are presented as mean values ± s.d. See Supplementary Fig. for additional biological replicates. Source data for Fig. 3c–g are provided as a Source Data file.

Article Snippet: Antibodies used were as follows: EXOSC8: Proteintech #11979-1-AP PRIM1: Cell Signaling Technology #4725 Vinculin: Sigma #V9131 See Source Data file for unprocessed scans of the most important blots.

Techniques: Positive Control, Disruption, Derivative Assay, Expressing, Sequencing, Infection, Western Blot

Journal: Cells

Article Title: Exosc9 Initiates SUMO-Dependent lncRNA TERRA Degradation to Impact Telomeric Integrity in Endocrine Therapy Insensitive Hormone Receptor-Positive Breast Cancer.

doi: 10.3390/cells12202495

Figure Lengend Snippet: Figure 1. Exosc9 is enriched at telomeres by interacting with SUMOylated HP1α. (A) Combined IF-FISH was conducted using Exosc9 antibody (green) and telomere probe (red) in MCF10-2A cells. The arrowheads indicate the colocalization of Exosc9 with telomeres. Scale bars: 10 µm (main images) and 1 µm (inset). IgG was used as a negative control to exclude the non-specific fluorescent signals. (B) Data from (A) were quantified and plotted. Data expressed as percentage ± SEM (Student’s t-test; *** p value < 0.001). (C) ChIP-qPCR was performed to confirm IF-FISH data for Exosc9 and to check for the presence of Exosc4 and Exosc10 at telomeres. IgG served as a negative control. ChIP values were normalized to mean % input and fold enrichment was compared to the IgG. Error bars correspond to mean ± SEM from three independent experiments. Student’s t-test was used to test for statistical significance. (D,E) Immobilized cell-free bait (unmodified and SUMOylated HP1α) captures prey (Exosc9). Western blot shows Exosc9 interaction with immunoprecipitated SUMO-modified (red arrows) and unmodified (black arrow) HP1α recombinant protein. (F) MCF10-2A cells were transfected with EV, wild-type HP1α (V5-HP1α wt), SUMO-deficient mutant HP1α (V5-SdHP1α), or SUMO3-HP1α (HA-SHP1α). ChIP of HP1α or EXOSC9. ChIP was quantified by qPCR to check whether HP1α SUMOylation is required; ChIP-qPCR was performed to assess the enrichment of HP1α and Exosc9 at telomeres. ChIP values were normalized to mean input, and fold enrichment was compared to the EV. The error bars correspond to mean ± SEM from three independent experiments. ANOVA and Tukey’s post-hoc tests were used to test for statistical significance. Transfection was validated by Western blot.

Article Snippet: Exoribonuclease assay was prepared using 1 μg of RNA in the reaction buffer (10 mM Tris HCL [PH 8.0], 10 mM DTT, Cells 2023, 12, 2495 5 of 16 50 mM KCL, 5 mM MgCl2, 1 U/μL RNase inhibitor) and incubated with/without EXOSC9 recombinant protein (Novus Biological) at 37 ◦C for different time periods (0 min, 15 min, 30 min, 45 min, and 60 min).

Techniques: Negative Control, ChIP-qPCR, Western Blot, Immunoprecipitation, Recombinant, Transfection, Mutagenesis

Journal: Cells

Article Title: Exosc9 Initiates SUMO-Dependent lncRNA TERRA Degradation to Impact Telomeric Integrity in Endocrine Therapy Insensitive Hormone Receptor-Positive Breast Cancer.

doi: 10.3390/cells12202495

Figure Lengend Snippet: Figure 2. Exosc9 is enriched at telomeres in a cell cycle-dependent manner. (A) MCF10-2A cells were synchronized using a double thymidine block. Cells were collected in the G1/S, S, and S/G2 phases. ChIP-qPCR was performed to assess the enrichment of Exosc9 at telomeres during the cell cycle. IgG served as a negative control. ChIP values were normalized to mean % input, and fold enrichment was compared between different cell cycle stages. Error bars correspond to mean ± SEM from three independent experiments. ANOVA and Tukey’s post-hoc tests were used to test for statistical significance. (B) MCF10-2A cells were synchronized using a double thymidine block. CB/IP was performed using HP1α for the IP. The input and IP samples were then immunoblotted for HP1α and Exosc9. Cyclin B1 was used as a marker for cell cycle synchronization. Red arrows distinguish between the IgG and HP1α bands. (C) MCF10-2A cells were synchronized using a double thymidine block. CB/IP was performed using HP1α for the IP. The input and IP samples were then immunoblotted for HP1α, SUMO2/3, and SENP7L. Cyclin B1 was used as a marker for cell cycle synchronization. The red arrows represent SUMO-conjugates of HP1α. (D,E) Blots were analyzed by densitometry using ImageJ, normalized to HP1α, and graphed as the mean ± SEM. ANOVA and Tukey’s post-hoc tests were used to test for statistical significance.

Article Snippet: Exoribonuclease assay was prepared using 1 μg of RNA in the reaction buffer (10 mM Tris HCL [PH 8.0], 10 mM DTT, Cells 2023, 12, 2495 5 of 16 50 mM KCL, 5 mM MgCl2, 1 U/μL RNase inhibitor) and incubated with/without EXOSC9 recombinant protein (Novus Biological) at 37 ◦C for different time periods (0 min, 15 min, 30 min, 45 min, and 60 min).

Techniques: Blocking Assay, ChIP-qPCR, Negative Control, Marker

Journal: Cells

Article Title: Exosc9 Initiates SUMO-Dependent lncRNA TERRA Degradation to Impact Telomeric Integrity in Endocrine Therapy Insensitive Hormone Receptor-Positive Breast Cancer.

doi: 10.3390/cells12202495

Figure Lengend Snippet: Figure 3. Exosc9 Drives TERRA Degradation. (A) MCF10-2A cells were transfected with nontargeting siRNA or Exosc9 siRNA.RT-PCR was carried out using primers corresponding to TERRA (1q-2q- 10q-13q), TERRA (15q), TERRA (Xp-Yp), XIST, HOTAIR, and fibronectin mRNA. CT values were normalized to β-actin, and fold change was compared to nontargeting siRNA. Error bars correspond to mean ± SEM from three independent experiments. Student’s t-test was used to test for statistical significance. (B) MCF10-2A cells were transfected with EV or OFPSpark-Exosc9. RT-PCR was carried out using primers corresponding to TERRA (1q-2q-10q-13q), TERRA (15q), TERRA (Xp-Yp), XIST, HOTAIR, and fibronectin mRNA. CT values were normalized to β-actin, and fold change was compared to EV. Error bars correspond to mean ± SEM from three independent experiments. Student’s t-test was used to test for statistical significance. In vitro, an exosomal activity assay was performed using 1 µg of RNA in a specific reaction buffer in the presence/absence of Exosc9 recombinant protein and was incubated at 37 ◦C. Samples from (0 min. 15 min, 30 min. 45 min, and 60 min) were analyzed using absolute-qPCR with (C) fibronectin-specific primers. (D) TERRA (15q)-specific primers. (E) TERRA (1q-2q-10q-13q)-specific primers. (F) TERRA (Xp-Yp)-specific primers. Error bars correspond to mean ± SEM from two independent experiments.

Article Snippet: Exoribonuclease assay was prepared using 1 μg of RNA in the reaction buffer (10 mM Tris HCL [PH 8.0], 10 mM DTT, Cells 2023, 12, 2495 5 of 16 50 mM KCL, 5 mM MgCl2, 1 U/μL RNase inhibitor) and incubated with/without EXOSC9 recombinant protein (Novus Biological) at 37 ◦C for different time periods (0 min, 15 min, 30 min, 45 min, and 60 min).

Techniques: Transfection, Reverse Transcription Polymerase Chain Reaction, In Vitro, Activity Assay, Recombinant, Incubation

Journal: Cells

Article Title: Exosc9 Initiates SUMO-Dependent lncRNA TERRA Degradation to Impact Telomeric Integrity in Endocrine Therapy Insensitive Hormone Receptor-Positive Breast Cancer.

doi: 10.3390/cells12202495

Figure Lengend Snippet: Figure 4. The Breast Invasive Carcinoma TCGA PanCancer Atlas dataset was utilized to identify luminal A breast cancer patients ((A), n = 499) who were treated with ((B), n = 286) or without ET (Supplemental Figure S5C, n = 211); specifically, ET treatments included tamoxifen, anastrazole, letrozole, exemestane, and/or fulvestrant. (C) The patient data were stratified to evaluate disease-free survival based on high versus low Exosc9 transcript levels; high and low quartile Exosc9 mRNA expression z-scores (relative to normal samples) were used for stratification. Exosc9 mRNA expression z-scores from the TCGA PanCancer Atlas samples were compared between luminal A breast cancer patients who received endocrine therapy and luminal A breast cancer patients who did not receive endocrine therapy. (D) Chromatin-bound protein immunoprecipitation for HP1α in TamS-7 (MCF7) and TamR-7 cells. Lysate and immunoprecipitation fractions were then immunoblotted for HP1α, SUMO2/3, and Exosc9. The red arrows represent SUMO-conjugates of HP1α. (E) SUMOylated-HP1α to total HP1α protein expression was compared, as well as (F) Exosc9 to total HP1α protein expression. (G) Tumor spheroids were formed from siRNA-treated TamR-7 cells. The white scale bar indicates 200µm in top two images and 50µm in bottom two images. Spheroid number (H) and spheroid diameter (I) were compared between siNT and siExosc9 transfected tumor spheroids. (Student’s t-test: * p value < 0.05; ** p value < 0.01).

Article Snippet: Exoribonuclease assay was prepared using 1 μg of RNA in the reaction buffer (10 mM Tris HCL [PH 8.0], 10 mM DTT, Cells 2023, 12, 2495 5 of 16 50 mM KCL, 5 mM MgCl2, 1 U/μL RNase inhibitor) and incubated with/without EXOSC9 recombinant protein (Novus Biological) at 37 ◦C for different time periods (0 min, 15 min, 30 min, 45 min, and 60 min).

Techniques: Expressing, Immunoprecipitation, Transfection

Journal: Cells

Article Title: Exosc9 Initiates SUMO-Dependent lncRNA TERRA Degradation to Impact Telomeric Integrity in Endocrine Therapy Insensitive Hormone Receptor-Positive Breast Cancer.

doi: 10.3390/cells12202495

Figure Lengend Snippet: Figure 5. Telo-FISH-stained metaphase chromosomes were evaluated from TamS-7 (MCF7) and TamR- 7 cells. (A) Representative images are included, which are characteristic of telomere morphology: normal, end-to-end fusion, single telomere loss, telomere at centromere, telomere amplification, and terminal deletion. White arrowheads highlight normal telomeres and yellow arrows indicate telomeric aberration.(B) Quantification of telomere morphology compared between TamS-7 and TamR-7. TERRA expression decreased in TamR-7 cells. (C) RT-PCR was performed on TERRA (1q-2q- 10q-13q), TERRA (15q), and TERRA (Xp-Yp), and normalized to β-actin in TamS-7 and TamR-7 cells. Fold change of TERRA expression was compared between TamS-7 and TamR-7 cells. Data represent the mean ± SEM of three independent experiments (Student’s t-test: ** p-value < 0.01). (D) DNA-RNA enrichment at the telomeres in Exosc9-deficient TamR-7 cells. TamR-7 cells were transfected with either siNT or siExosc9. Chromatin immunoprecipitation was performed using the S9.6 antibody, and enrichment at the telomeres was compared. Data represent the mean ± SEM of three independent experiments (Student’s t-test: * p-value < 0.05). (E) TamR-7 cells were transfected with siRNA-siNT or siExosc9. Cells were then fixed and chromatin immunoprecipitated for γH2Ax. Telomere enrichment was compared to the IgG control (Student’s t-test, ** p-value < 0.01). (F) Noncancerous mammary epithelial cells were treated with either empty vector or overexpression for Exosc9. Cells were then fixed and chromatin immunoprecipitated for γH2Ax. Telomere enrichment was compared to the IgG control. (Student’s t-test, * p-value < 0.05).

Article Snippet: Exoribonuclease assay was prepared using 1 μg of RNA in the reaction buffer (10 mM Tris HCL [PH 8.0], 10 mM DTT, Cells 2023, 12, 2495 5 of 16 50 mM KCL, 5 mM MgCl2, 1 U/μL RNase inhibitor) and incubated with/without EXOSC9 recombinant protein (Novus Biological) at 37 ◦C for different time periods (0 min, 15 min, 30 min, 45 min, and 60 min).

Techniques: Staining, Expressing, Reverse Transcription Polymerase Chain Reaction, Transfection, Chromatin Immunoprecipitation, Immunoprecipitation, Control, Plasmid Preparation, Over Expression

Journal: ACS Applied Bio Materials

Article Title: Hydrogel-Based Extracellular Vesicle Isolation Method from Various Biological Solutions

doi: 10.1021/acsabm.5c01838

Figure Lengend Snippet: Cellular uptake of isolated MSC-derived EVs isolated using the HAS method in different cell types. (A) Internalization of EVs in dermal papilla cells. EV membranes were labeled with ExoSparkler EX01 (green) and cell nuclei were stained with DAPI (blue). (B) Internalization of EVs in skin cells. EV proteins were labeled with ExoSparkler EX05 (red), and cell nuclei and cytoskeleton were stained with DAPI (blue) and phalloidin (green).

Article Snippet: Proteins within MSC-EVs were labeled with ExoSparkler EX05 (Dojindo Laboratories).

Techniques: Isolation, Derivative Assay, Labeling, Staining

Journal: Acta Neuropathologica

Article Title: Biomarkers for parkinsonian disorders in CNS-originating EVs: promise and challenges

doi: 10.1007/s00401-023-02557-1

Figure Lengend Snippet: Summary of EV-associated miRNAs in Parkinson’s disease

Article Snippet: Plasma , PD = 52 HC = 48 , PureExo Exosome Isolation (101Bio) , Exosomal RNA and Protein Extraction kit (101Bio) , qRT-PCR , miR-626, miR-505, miR-181c, miR-331-5p, miR-193-p, miR-196-p, miR-454, miR-125a-p, miR-137 , miR-331-5p , miR-505 , d AUC = 0.898 , [ ] .

Techniques: Isolation, RNA Extraction, TaqMan microRNA Assay, RNA Sequencing, Clinical Proteomics, Protein Extraction, Next-Generation Sequencing, Immunoprecipitation, Microarray, Hybridization

Journal: Scientific Reports

Article Title: Effect of nHA/CS/PLGA delivering adipose stem cell-derived exosomes and bone marrow stem cells on bone healing—in vitro and in vivo studies

doi: 10.1038/s41598-024-76672-8

Figure Lengend Snippet: Materials and instruments used in methods.

Article Snippet: Exosomal Protein Extraction Kit , FUJIFILM Wako Chemicals U.S.A. Corporation, Richmond, VA, USA.

Techniques: Staining, Protein Extraction, ALP Assay, Lysis, Bicinchoninic Acid Protein Assay, Paraffin Wax, H&E Stain