Journal: Journal of Hematology & Oncology

Article Title: An Auristatin nanoconjugate targeting CXCR4+ leukemic cells blocks acute myeloid leukemia dissemination

doi: 10.1186/s13045-020-00863-9

Figure Lengend Snippet: Antineoplastic activity of T22-GFP-H6-Auristatin in a disseminated AML mouse model. a Experimental design of the in vivo assay used to assess the antineoplastic activity of T22-GFP-H6-Auristatin. b Measurement of total body weight of mice during the experimental time according to the treatment group. Results are presented as mean ± SE body weight in grams. c Comparison of evolution of bioluminescence emission in mice treated with vehicle (VEH) or T22-GFP-H6-Auristatin (T22-AUR) and untreated healthy mice (Normal group) the days 4, 8, 11, and 13 after injection of THP-1-Luci cells, measured by the IVIS Spectrum. d Follow-up of total body bioluminescence emission of mice treated with T22-GFP-H6-Auristatin (T22-AUR) or vehicle (VEH) and untreated healthy mice (Normal) during all the experiment, analyzed in IVIS Spectrum. Results are presented as mean ± SE luminescence values in photons per second (total flux [p/s]). U of Mann-Whitney test was used to assess significant differences between groups in these studies ( b , d ), and these differences were considered statistically significant when the p value was lower than 0.05. * indicates differences between the VEH and T22-AUR groups, # between the Normal and VEH groups, and ‡ between the Normal and T22-AUR groups. BLI, bioluminescence; T22-AUR, T22-GFP-H6-Auristatin group; VEH, vehicle group.;SE, standard error

Article Snippet: The volume size distribution of T22-GFP-H6 nanoparticles and resulting nanoconjugates (T22-GFP-H6-Auristatin) was determined by dynamic light scattering at 633 nm in a Zetasizer Nano (Malvern Instruments, Malvern, UK).

Techniques: Activity Assay, In Vivo, Comparison, Injection, MANN-WHITNEY

Journal: Journal of Hematology & Oncology

Article Title: An Auristatin nanoconjugate targeting CXCR4+ leukemic cells blocks acute myeloid leukemia dissemination

doi: 10.1186/s13045-020-00863-9

Figure Lengend Snippet: T22-GFP-H6-Auristatin nanoconjugates characterization and stability. a T22-GFP-H6 nanoparticle conjugation with maleimide functionalized Monomethyl Auristatin E (MMAE). b MALDI-TOF mass spectrometry of wild-type T22-GFP-H6 nanoparticles and T22-GFP-H6-Auristatin nanoconjugates (top) and free MMAE before and after nanoconjugates re-purification (bottom). Each peak corresponds to the covalent addition (+ 911 Da) of a single MMAE molecule (top). Red boxes indicate free Auristatin mass spectrometry spectra (bottom). c Hydrodynamic volume size distribution of wild-type T22-GFP-H6 nanoparticles (red) and T22-GFP-H6-Auristatin nanoconjugates (blue) determined by dynamic light scattering. Samples were analyzed in triplicate and data represented as mean +/− SE. PDI indicates polydispersion index. d Average molar mass distribution of wild-type T22-GFP-H6 nanoparticles (red) and T22-GFP-H6-Auristatin nanoconjugates (blue) determined by size exclusion chromatography coupled to multi-angle light scattering (SEC-MALS). e Representative electron microscopy (FESEM) images of T22-GFP-H6-Auristatin nanoconjugates presented in two magnifications (see inset). Scale bars indicate 100 nm. In the bottom, the quantitative average size of nanoconstructs determined by image analysis and shown as mean ± SE. f Proteolytic stability of T22-GFP-H6-Auristatin nanoconjugates in human serum at different incubation times up to 24 h analyzed by western blot immunodetection with a monoclonal anti-His antibody. “S” indicates human serum control. MW, molecular weight; SE, standard error

Article Snippet: The volume size distribution of T22-GFP-H6 nanoparticles and resulting nanoconjugates (T22-GFP-H6-Auristatin) was determined by dynamic light scattering at 633 nm in a Zetasizer Nano (Malvern Instruments, Malvern, UK).

Techniques: Conjugation Assay, Mass Spectrometry, Purification, Size-exclusion Chromatography, Multi-Angle Light Scattering, Electron Microscopy, Incubation, Western Blot, Immunodetection, Control, Molecular Weight

Journal: Journal of Hematology & Oncology

Article Title: An Auristatin nanoconjugate targeting CXCR4+ leukemic cells blocks acute myeloid leukemia dissemination

doi: 10.1186/s13045-020-00863-9

Figure Lengend Snippet: Internalization capacity and antineoplastic activity of T22-GFP-H6 and T22-GFP-H6-Auristatin in AML cell lines. a CXCR4 membrane expression of THP-1 and SKM-1 AML cell lines by flow cytometry. b Evaluation of T22-GFP-H6-Auristatin internalization, by flow cytometry, in CXCR4+ leukemic cells after 1 h of incubation at different concentrations. c Competition assay with AMD3100 (240 nM T22-GFP-H6-Auristatin and 2000 nM AMD3100) to determine the specificity of internalization through CXCR4 receptor. d Evaluation of antineoplastic activity of T22-GFP-H6 after 48 h of treatment in AML cell lines performed by XTT assay. e Anticancer activity of T22-GFP-H6-Auristatin in AML cell lines after 48 h of incubation by XTT assay. f Competition of antineoplastic activity of T22-GFP-H6-Auristatin at 240 nM with AMD3100 at 2000 nM after 48 h exposure by XTT assay. Results are presented as mean ± SE MFI in a , b , and c , and mean ± SE percent of cell viability in d , e, and f . U of Mann-Whitney test was used to test differences between groups. In b and e , T22-GFP-H6-Auristatin treated cells were compared with the buffer treated cells (Ctrl). Statistical significant differences are indicated by * when p value < 0.05 and ** when p value < 0.001. n.s. means no significance. AML, acute myeloid leukemia; Ctrl, control; MFI, mean fluorescence intensity; SE, standard error

Article Snippet: The volume size distribution of T22-GFP-H6 nanoparticles and resulting nanoconjugates (T22-GFP-H6-Auristatin) was determined by dynamic light scattering at 633 nm in a Zetasizer Nano (Malvern Instruments, Malvern, UK).

Techniques: Activity Assay, Membrane, Expressing, Flow Cytometry, Incubation, Competitive Binding Assay, XTT Assay, MANN-WHITNEY, Control, Fluorescence

Journal: Journal of Hematology & Oncology

Article Title: An Auristatin nanoconjugate targeting CXCR4+ leukemic cells blocks acute myeloid leukemia dissemination

doi: 10.1186/s13045-020-00863-9

Figure Lengend Snippet: Study of cell death in AML cell lines caused by T22-GFP-H6-Auristatin treatment. Study of the mechanism of T22-GFP-H6-Auristatin-induced cell death. a Induction of apoptosis and mitotic catastrophe by T22-GFP-H6-Auristatin at 240 nM for 24 and 48 h of exposure detected by DAPI staining in CXCR4-positive THP-1 and SKM-1 leukemic cell lines. Yellow and red arrows point to MC figures and apoptotic bodies, respectively. b Quantitation of the number of cells undergoing mitotic catastrophe triggered by treatment with T22-GFP-H6-Auristatin at 240 nM in THP-1 and SKM-1 for 24 (orange bars) or 48 h (yellow bars). c Quantitation of the number of apoptotic bodies in THP-1 and SKM-1 induced by exposure to 240 nM T22-GFP-H6-Auristatin for 24 (orange bars) or 48 h (yellow bars). Results in b and c are presented as mean ± SE cell number for each cell death event (MC or apoptosis) in all fields analyzed. * indicates significant differences between groups with a p value < 0.05. CTRL, control; MC, mitotic catastrophe; SE, standard error

Article Snippet: The volume size distribution of T22-GFP-H6 nanoparticles and resulting nanoconjugates (T22-GFP-H6-Auristatin) was determined by dynamic light scattering at 633 nm in a Zetasizer Nano (Malvern Instruments, Malvern, UK).

Techniques: Staining, Quantitation Assay, Control

Journal: Journal of Hematology & Oncology

Article Title: An Auristatin nanoconjugate targeting CXCR4+ leukemic cells blocks acute myeloid leukemia dissemination

doi: 10.1186/s13045-020-00863-9

Figure Lengend Snippet: Bioluminescence intensity levels emitted in different body sections of mice along the experimental time

Article Snippet: The volume size distribution of T22-GFP-H6 nanoparticles and resulting nanoconjugates (T22-GFP-H6-Auristatin) was determined by dynamic light scattering at 633 nm in a Zetasizer Nano (Malvern Instruments, Malvern, UK).

Techniques: Injection

Journal: Journal of Hematology & Oncology

Article Title: An Auristatin nanoconjugate targeting CXCR4+ leukemic cells blocks acute myeloid leukemia dissemination

doi: 10.1186/s13045-020-00863-9

Figure Lengend Snippet: Antineoplastic activity of T22-GFP-H6-Auristatin in the bone marrow, circulating blood, and affected extramedullar sites ex vivo. a Comparison of bioluminescence emission between the T22-GFP-H6-Auristatin-treated (T22-AUR, orange bars), vehicle-treated (VEH, blue bars), and untreated healthy mice (Normal, green bars) groups in the bone marrow, liver, and spleen. b Bioluminescence Images comparing leukemia dissemination in the bone marrow, liver, and spleen of mice treated with the nanoconjugate (T22-AUR) or vehicle (VEH), and untreated healthy mice (Normal). c Comparison of levels of bioluminescence emitted by circulating blood between groups (VEH vs T22-AUR) detected ex vivo at the end of the experiment. d Detection of CD45-positive cells in the peripheral blood by flow cytometry in mice treated with vehicle (VEH, blue) or T22-GFP-H6-Auristatin (T22-AUR, orange). Results are presented as percentage, normalizing the number of CD45-positive cells found in a sample by the mean of CD45-positive cells found in all vehicle-treated mice. e Histogram and dot plot representations of FL3-H fluorescence detection in a mouse blood sample treated with vehicle (VEH, blue) overlapped with another treated with T22-GFP-H6-Auristatin (T22-AUR, orange). M1 and R2 sections indicate CD45-positive cells, whereas R1 section indicates CD45-negative cells. Results in studies a and c are presented as mean ± SE luminescence values in photons per second (total flux [p/s]), and as mean ± SE of the percent of CD45-positive cells in d . U of Mann-Whitney test was used in studies a , c , and d to assess significant differences and are presented in the figures with an * to compare between the VEH and T22-AUR groups, # between the Normal and VEH groups, and ‡ between the Normal and T22-AUR groups. The differences between groups were considered significant when the p value was lower than 0.05. PB, peripheral blood; T22-AUR, T22-GFP-H6-Auristatin group; VEH, vehicle group; SE, standard error

Article Snippet: The volume size distribution of T22-GFP-H6 nanoparticles and resulting nanoconjugates (T22-GFP-H6-Auristatin) was determined by dynamic light scattering at 633 nm in a Zetasizer Nano (Malvern Instruments, Malvern, UK).

Techniques: Activity Assay, Ex Vivo, Comparison, Flow Cytometry, Fluorescence, MANN-WHITNEY

Journal: Journal of Hematology & Oncology

Article Title: An Auristatin nanoconjugate targeting CXCR4+ leukemic cells blocks acute myeloid leukemia dissemination

doi: 10.1186/s13045-020-00863-9

Figure Lengend Snippet: Detection of CD45-positive cells in the leukemia-infiltrated tissues after treatment with T22-GFP-H6-Auristatin. a Detection of CD45-positive cells by IHC in the bone marrow and extramedullar organs (spleen and liver) of mice treated with vehicle (VEH) or T22-GFP-H6-Auristatin (T22-AUR) 14 days after injection of CXCR4+ THP-1-Luci cells, and comparison with CD45-positive cell detection in same tissues of a normal non-leukemic mouse. b Quantification of the CD45 detection by IHC in the spleen, liver, and bone marrow in mice treated with vehicle (VEH, blue bars) and T22-GFP-H6-Auristatin (T22-AUR, orange bars). This quantification is presented as mean ± SE percentage of stained surface with CD45 antibody in the tissue of all mice. c Quantitation of CD45 staining intensity by IHC in the liver, spleen, and bone marrow in mice treated with vehicle (VEH, blue bars) or T22-GFP-H6-Auristatin (T22-AUR, orange bars). Results are presented as the mean ± SE of the mean gray value obtained in Image J (see the “ ” section). Significant differences were indicated by * when the p value was lower than 0.05, using U of Mann-Whitney test between groups. T22-AUR, T22-GFP-H6-Auristatin group; VEH, vehicle group; SE, standard error

Article Snippet: The volume size distribution of T22-GFP-H6 nanoparticles and resulting nanoconjugates (T22-GFP-H6-Auristatin) was determined by dynamic light scattering at 633 nm in a Zetasizer Nano (Malvern Instruments, Malvern, UK).

Techniques: Injection, Comparison, Staining, Quantitation Assay, MANN-WHITNEY

Journal: Journal of Hematology & Oncology

Article Title: An Auristatin nanoconjugate targeting CXCR4+ leukemic cells blocks acute myeloid leukemia dissemination

doi: 10.1186/s13045-020-00863-9

Figure Lengend Snippet: Hematoxylin and eosin staining in leukemia-infiltrated tissues after treatment with T22-GFP-H6-Auristatin. Hematoxylin and eosin staining of the liver, spleen, bone marrow, lung, pancreas, brain, heart, and kidneys of mice treated with vehicle (VEH) or T22-GFP-H6-Auristatin (T22-AUR) 14 days after the injection of CXCR4+ THP-1-Luci cells in NSG mice, compared with the stained tissues of a healthy mouse (Normal). T22-AUR, T22-GFP-H6-Auristatin group; VEH, vehicle group

Article Snippet: The volume size distribution of T22-GFP-H6 nanoparticles and resulting nanoconjugates (T22-GFP-H6-Auristatin) was determined by dynamic light scattering at 633 nm in a Zetasizer Nano (Malvern Instruments, Malvern, UK).

Techniques: Staining, Injection

Journal: Journal of Extracellular Biology

Article Title: A One‐Step Workflow for Size‐Based Separation of Extracellular Vesicles With Integrated Surface Marker Detection

doi: 10.1002/jex2.70109

Figure Lengend Snippet: Representation and validation of the AF4‐MALS‐FLD method . (A) Overview of the workflow used for identification of EV surface proteins. PE‐conjugated antibodies were incubated with the sample (e.g. pre‐purified EVs, cell culture supernatant, urine, or plasma) and loaded into the AF4 channel. (B) The light scatter elution profile (in relative scale) (black, full line), UV elution profile (black, dotted line) and the size determination ( R rms in nm) (red) obtained by the multi‐angle light scattering (MALS) detector is plotted against time for labelling of SK‐BR‐3‐derived EVs with PE‐conjugated anti‐CD81 antibody. (C) The fluorescent light detector (FLD) signal (in relative scale) for SK‐BR‐3‐derived EVs labelled with PE‐conjugated anti‐CD9, anti‐CD63 and anti‐CD81 is plotted against time. (D) Transmission electron microscopy (TEM) images of different fractions of the AF4‐MALS‐FLD elution profile are shown (scale bar = 200 nm).

Article Snippet: The following primary and secondary antibodies were used for western blot analysis: mouse monoclonal anti‐Alix (1:1000) (cat no. 2171S, Cell Signaling Technology), rabbit monoclonal anti‐CD9 (1:1000) (cat no. 13403S, Cell Signaling Technology), rabbit monoclonal anti‐Syntenin‐1 (1:1000) (cat no. ab133267, Abcam), mouse monoclonal anti‐TSG101 (1:1000) (cat no. sc‐7964, Santa Cruz Biotechnology), rabbit monoclonal anti‐PSMA (1:1000) (cat no. 12702S), mouse monoclonal anti‐EpCAM (1:1000) (cat no. 2929S, Cell Signaling Technology), rabbit monoclonal anti‐HER2 (1:1000) (cat no. 2165S, Cell Signaling Technology), mouse monoclonal anti‐GAPDH (1:2500) (cat no. G8795, Merck Life Science), sheep anti‐mouse horseradish peroxidase‐linked (1:3000) (cat no. NA931V, GE Healthcare Life Sciences) and donkey anti‐rabbit horseradish peroxidase‐linked antibody (1:8000) (cat no. NA934V, GE Healthcare Life Sciences).

Techniques: Biomarker Discovery, Incubation, Purification, Cell Culture, Clinical Proteomics, Multi-Angle Light Scattering, Derivative Assay, Transmission Assay, Electron Microscopy

Journal: Journal of Extracellular Biology

Article Title: A One‐Step Workflow for Size‐Based Separation of Extracellular Vesicles With Integrated Surface Marker Detection

doi: 10.1002/jex2.70109

Figure Lengend Snippet: AF4‐MALS‐FLD analysis of EV surface proteins with biomarker potential in prostate and breast cancer . MCF‐7‐, MDA‐MB‐231‐ and SK‐BR‐3‐derived EVs were labelled with PE‐conjugated anti‐EpCAM antibodies and analysed by AF4‐MALS‐FLD. (A) The elution profile (in relative scale) of the multi‐angle light scatter (MALS) detector and the size ( R rms in nm) were plotted against time. The fluorescent light detector (FLD) signal for MCF‐7‐, MDA‐MB‐231‐ and SK‐BR‐3‐derived EVs labelled with (B) PE‐conjugated anti‐EpCAM and (C) PE‐conjugated anti‐HER2 antibodies were plotted. (D) From FLD elution profiles, the area under the curve for the EV peak (24–80 min) was determined. Unstained EV samples were used as a negative control. (E) Different concentrations (6 × 10 9 , 8 × 10 9 , 1 × 10 10 and 2 × 10 10 particles as measured by NTA) including a negative control of LNCaP‐derived EVs (high PSMA expression) were labelled with anti‐PSMA antibodies and analysed by the AF4‐MALS‐FLD protocol. (F) The area under the curve for the EV peak was determined for LNCaP‐derived EVs. Different concentrations (2 × 10 10 , 4 × 10 10 and 6 × 10 10 particles as measured by NTA) including a negative control of (G) MCF‐7‐derived EVs (high EpCAM expression) or (I) SK‐BR‐3‐derived EVs (high HER2 expression) were labelled with PE‐conjugated anti‐EpCAM or anti‐HER2 antibodies respectively and analysed by the AF4‐MALS‐FLD protocol. The area under the curve for the EV peak (24–80 min) was determined for (H) MCF‐7‐ and (J) SK‐BR‐3‐derived EVs.

Article Snippet: The following primary and secondary antibodies were used for western blot analysis: mouse monoclonal anti‐Alix (1:1000) (cat no. 2171S, Cell Signaling Technology), rabbit monoclonal anti‐CD9 (1:1000) (cat no. 13403S, Cell Signaling Technology), rabbit monoclonal anti‐Syntenin‐1 (1:1000) (cat no. ab133267, Abcam), mouse monoclonal anti‐TSG101 (1:1000) (cat no. sc‐7964, Santa Cruz Biotechnology), rabbit monoclonal anti‐PSMA (1:1000) (cat no. 12702S), mouse monoclonal anti‐EpCAM (1:1000) (cat no. 2929S, Cell Signaling Technology), rabbit monoclonal anti‐HER2 (1:1000) (cat no. 2165S, Cell Signaling Technology), mouse monoclonal anti‐GAPDH (1:2500) (cat no. G8795, Merck Life Science), sheep anti‐mouse horseradish peroxidase‐linked (1:3000) (cat no. NA931V, GE Healthcare Life Sciences) and donkey anti‐rabbit horseradish peroxidase‐linked antibody (1:8000) (cat no. NA934V, GE Healthcare Life Sciences).

Techniques: Biomarker Discovery, Derivative Assay, Multi-Angle Light Scattering, Negative Control, Expressing

Journal: Journal of Extracellular Biology

Article Title: A One‐Step Workflow for Size‐Based Separation of Extracellular Vesicles With Integrated Surface Marker Detection

doi: 10.1002/jex2.70109

Figure Lengend Snippet: Detection of EVs in complex matrices . (A) Different volumes of cell culture supernatant (0, 20, 40 and 60 µL) collected from the MCF‐7 cells were labelled with PE‐conjugated anti‐EpCAM antibodies and analysed by AF4‐MALS‐FLD. The area under the curve for the EV peak in complex matrices (40–80 min) was determined. (B) Different amounts of LNCaP‐derived EVs were spiked in 100 µL of concentrated urine, diluted 1:1 in PBS to reduce viscosity, labelled with PE‐conjugated anti‐PSMA antibodies, and analysed by AF4‐MALS‐FLD. The area under the curve for the EV peak was determined. Different amounts of (C) MCF‐7‐ or (D) SK‐BR‐3‐derived EVs were spiked in 100 µL of blood plasma, diluted 1:1 in PBS to reduce viscosity, and labelled with PE‐conjugated anti‐EpCAM or anti‐HER2 antibodies, respectively. Labelled EVs were analysed by AF4‐MALS‐FLD and the area under the curve for the EV peak was determined. Different amounts of SK‐BR‐3 EVs were also spiked in blood plasma and labelled with isotype control antibodies. (E) Different concentrations of soluble EpCAM (1, 5 and 10 ng/mL) and soluble HER2 (50, 100 and 150 ng/mL) were spiked in blood plasma, labelled with PE‐conjugated anti‐EpCAM or anti‐HER2 antibodies respectively, and analysed by AF4‐MALS‐FLD.

Article Snippet: The following primary and secondary antibodies were used for western blot analysis: mouse monoclonal anti‐Alix (1:1000) (cat no. 2171S, Cell Signaling Technology), rabbit monoclonal anti‐CD9 (1:1000) (cat no. 13403S, Cell Signaling Technology), rabbit monoclonal anti‐Syntenin‐1 (1:1000) (cat no. ab133267, Abcam), mouse monoclonal anti‐TSG101 (1:1000) (cat no. sc‐7964, Santa Cruz Biotechnology), rabbit monoclonal anti‐PSMA (1:1000) (cat no. 12702S), mouse monoclonal anti‐EpCAM (1:1000) (cat no. 2929S, Cell Signaling Technology), rabbit monoclonal anti‐HER2 (1:1000) (cat no. 2165S, Cell Signaling Technology), mouse monoclonal anti‐GAPDH (1:2500) (cat no. G8795, Merck Life Science), sheep anti‐mouse horseradish peroxidase‐linked (1:3000) (cat no. NA931V, GE Healthcare Life Sciences) and donkey anti‐rabbit horseradish peroxidase‐linked antibody (1:8000) (cat no. NA934V, GE Healthcare Life Sciences).

Techniques: Cell Culture, Derivative Assay, Viscosity, Clinical Proteomics, Control

Journal: Journal of Extracellular Biology

Article Title: A One‐Step Workflow for Size‐Based Separation of Extracellular Vesicles With Integrated Surface Marker Detection

doi: 10.1002/jex2.70109

Figure Lengend Snippet: Validation of the AF4‐MALS‐FLD workflow on patient samples . Urine samples of five prostate cancer patients were labelled for PSMA and analysed by the AF4‐MALS‐FLD workflow. Fractions 40–80 min were collected, concentrated and processed for mass spectrometry‐based proteomic analysis. (A) EV markers Syntenin‐1, Flotillin‐1, CD63, CD9, CD81, Flotillin‐2, Alix and TSG101 were analysed (missing sample indicated in grey). Z ‐score transformation of intensities were plotted. (B) Targeted mass spectrometry analysed the presence of PSMA (FOLH1) in patient samples. The z ‐score transformation of intensities was plotted with the AF4‐MALS‐FLD peak area. (C) Blood plasma samples of healthy controls ( n = 7) and HER2 amplified breast cancer patients ( n = 10) were labelled with PE‐conjugated anti‐HER2 antibodies. (D) Blood plasma samples of healthy controls ( n = 6) and breast cancer patients ( n = 8) were labelled with PE‐conjugated anti‐EpCAM antibodies. The area under the curve values were normalised for the mean value in the healthy control group.

Article Snippet: The following primary and secondary antibodies were used for western blot analysis: mouse monoclonal anti‐Alix (1:1000) (cat no. 2171S, Cell Signaling Technology), rabbit monoclonal anti‐CD9 (1:1000) (cat no. 13403S, Cell Signaling Technology), rabbit monoclonal anti‐Syntenin‐1 (1:1000) (cat no. ab133267, Abcam), mouse monoclonal anti‐TSG101 (1:1000) (cat no. sc‐7964, Santa Cruz Biotechnology), rabbit monoclonal anti‐PSMA (1:1000) (cat no. 12702S), mouse monoclonal anti‐EpCAM (1:1000) (cat no. 2929S, Cell Signaling Technology), rabbit monoclonal anti‐HER2 (1:1000) (cat no. 2165S, Cell Signaling Technology), mouse monoclonal anti‐GAPDH (1:2500) (cat no. G8795, Merck Life Science), sheep anti‐mouse horseradish peroxidase‐linked (1:3000) (cat no. NA931V, GE Healthcare Life Sciences) and donkey anti‐rabbit horseradish peroxidase‐linked antibody (1:8000) (cat no. NA934V, GE Healthcare Life Sciences).

Techniques: Biomarker Discovery, Mass Spectrometry, Transformation Assay, Clinical Proteomics, Amplification, Control

Journal: bioRxiv

Article Title: Physical, chemical, and structural properties of human gastric organoid-derived mucus

doi: 10.1101/2025.11.08.687257

Figure Lengend Snippet: ( A ) Alcian blue staining for acidic mucins on tissue surface epithelium (top) and in the organoid lumen (bottom). Representative of 3 experiments. ( B ) Immunofluorescence staining for MUC5AC (green) on the surface epithelium and in the pit regions of human gastric tissue (top) and in the organoid lumen (bottom). Representative of two experiments. ( C ) Immunofluorescence staining for MUC6 in the neck regions of the gastric glands and on the outer edges of the organoid lumen. Representative of 2 experiments. All scale bars = 200 μm.

Article Snippet: Sections were blocked for 30 min with animal-free blocking reagent (Vector Laboratories) and incubated with primary antibodies to E-cadherin (Biolegend Cat. 324102), MUC5AC (Santa Cruz Biotech Cat. 398985), and MUC6 (Invitrogen Cat. PA5-50557) overnight at 4°C.

Techniques: Staining, Immunofluorescence

Journal: bioRxiv

Article Title: Physical, chemical, and structural properties of human gastric organoid-derived mucus

doi: 10.1101/2025.11.08.687257

Figure Lengend Snippet: (A ) Timeline for 2D culture conditions, mucus collection, and functional analyses. ( B ) Time-dependent development of transepithelial electrical resistance (TEER); one representative out of seven experiments with four technical replicates. Mean ± SD; dotted line indicates TEER threshold for airlift at y=200 Ωcm 2 . ( C ) Schematic diagram of Transwell 2D culture conditions: submerged (left) and air-liquid interface, ALI (right). ( D ) Clear, viscoelastic mucus on the Transwell insert (left) and removed from the apical epithelium (right). ( E ) Bioengineered mucus (BGM) dry mass collected per well every other day (mean ± SD), representative of two experiments with 3-5 technical replicates. Dry mass was determined by weighing lyophilized samples. ( F ) Concentration (mg dry weight per mL) of BGM (n=23), native mucus (n=6), and L-WRN culture media (n=8). Data analyzed by one-way ANOVA with Tukey’s multiple comparisons test; **** P ≤ 0.0001. ( G ) Concentration of MUC5AC in BGM (n=10) and NM (n=5) determined by ELISA. ( H ) Size exclusion chromatography paired with multi-angle light-scattering (SEC-MALS) analysis of BGM (top) and NM (bottom) showing the presence of low and high molecular weight compounds based on differential refractive indices (dRI) in blue and the light scattering (LS) signal in red. ( I ) Molecular weights of a porcine gastric mucin (PGM) control, BGM (n=3), and NM (n=4) determined by SEC-MALS. Data analyzed by one-way ANOVA with Tukey’s multiple comparisons test. All bar graphs include individual data points, mean ± SD.

Article Snippet: Sections were blocked for 30 min with animal-free blocking reagent (Vector Laboratories) and incubated with primary antibodies to E-cadherin (Biolegend Cat. 324102), MUC5AC (Santa Cruz Biotech Cat. 398985), and MUC6 (Invitrogen Cat. PA5-50557) overnight at 4°C.

Techniques: Functional Assay, Concentration Assay, Enzyme-linked Immunosorbent Assay, Size-exclusion Chromatography, Multi-Angle Light Scattering, High Molecular Weight, Control

Journal: bioRxiv

Article Title: Physical, chemical, and structural properties of human gastric organoid-derived mucus

doi: 10.1101/2025.11.08.687257

Figure Lengend Snippet: Three biological replicates of BGM, four native human gastric mucus samples collected from surgical material, and one porcine gastric mucin sample were analyzed by LC/MC. ( A ) Number of proteins identified by mass spectrometry in bioengineered gastric mucus (BGM; n=3), native mucus (NM; n=4), and porcine gastric mucin (PGM; n=1) samples. ( B ) Number of total and shared proteins (overlapping region) between BGM and NM (top) and BGM and PGM (bottom). ( C ) Relative abundance of gastrointestinal mucins MUC5AC, 1, 6, and 2 and trefoil factors (TFF) 1 and 2 in BGM (blue), NM (red), and PGM (grey). Combined data from all identified protein isotypes. ( D ) Categorization of proteins detected in PGM (left) and BGM (right) based on protein functions and cellular distribution listed in the Human Protein Atlas . Percentage values represent cumulative relative protein intensity values for each category. ( E ) Heatmap showing relative expression of key stomach-specific factors identified in BGM and NM. Data from n=4 NM and n=3 BGM samples.

Article Snippet: Sections were blocked for 30 min with animal-free blocking reagent (Vector Laboratories) and incubated with primary antibodies to E-cadherin (Biolegend Cat. 324102), MUC5AC (Santa Cruz Biotech Cat. 398985), and MUC6 (Invitrogen Cat. PA5-50557) overnight at 4°C.

Techniques: Mass Spectrometry, Expressing

Journal: Journal of Extracellular Biology

Article Title: A One‐Step Workflow for Size‐Based Separation of Extracellular Vesicles With Integrated Surface Marker Detection

doi: 10.1002/jex2.70109

Figure Lengend Snippet: Representation and validation of the AF4‐MALS‐FLD method . (A) Overview of the workflow used for identification of EV surface proteins. PE‐conjugated antibodies were incubated with the sample (e.g. pre‐purified EVs, cell culture supernatant, urine, or plasma) and loaded into the AF4 channel. (B) The light scatter elution profile (in relative scale) (black, full line), UV elution profile (black, dotted line) and the size determination ( R rms in nm) (red) obtained by the multi‐angle light scattering (MALS) detector is plotted against time for labelling of SK‐BR‐3‐derived EVs with PE‐conjugated anti‐CD81 antibody. (C) The fluorescent light detector (FLD) signal (in relative scale) for SK‐BR‐3‐derived EVs labelled with PE‐conjugated anti‐CD9, anti‐CD63 and anti‐CD81 is plotted against time. (D) Transmission electron microscopy (TEM) images of different fractions of the AF4‐MALS‐FLD elution profile are shown (scale bar = 200 nm).

Article Snippet: The following primary and secondary antibodies were used for western blot analysis: mouse monoclonal anti‐Alix (1:1000) (cat no. 2171S, Cell Signaling Technology), rabbit monoclonal anti‐CD9 (1:1000) (cat no. 13403S, Cell Signaling Technology), rabbit monoclonal anti‐Syntenin‐1 (1:1000) (cat no. ab133267, Abcam), mouse monoclonal anti‐TSG101 (1:1000) (cat no. sc‐7964, Santa Cruz Biotechnology), rabbit monoclonal anti‐PSMA (1:1000) (cat no. 12702S), mouse monoclonal anti‐EpCAM (1:1000) (cat no. 2929S, Cell Signaling Technology), rabbit monoclonal anti‐HER2 (1:1000) (cat no. 2165S, Cell Signaling Technology), mouse monoclonal anti‐GAPDH (1:2500) (cat no. G8795, Merck Life Science), sheep anti‐mouse horseradish peroxidase‐linked (1:3000) (cat no. NA931V, GE Healthcare Life Sciences) and donkey anti‐rabbit horseradish peroxidase‐linked antibody (1:8000) (cat no. NA934V, GE Healthcare Life Sciences).

Techniques: Biomarker Discovery, Incubation, Purification, Cell Culture, Clinical Proteomics, Multi-Angle Light Scattering, Derivative Assay, Transmission Assay, Electron Microscopy

Journal: Journal of Extracellular Biology

Article Title: A One‐Step Workflow for Size‐Based Separation of Extracellular Vesicles With Integrated Surface Marker Detection

doi: 10.1002/jex2.70109

Figure Lengend Snippet: AF4‐MALS‐FLD analysis of EV surface proteins with biomarker potential in prostate and breast cancer . MCF‐7‐, MDA‐MB‐231‐ and SK‐BR‐3‐derived EVs were labelled with PE‐conjugated anti‐EpCAM antibodies and analysed by AF4‐MALS‐FLD. (A) The elution profile (in relative scale) of the multi‐angle light scatter (MALS) detector and the size ( R rms in nm) were plotted against time. The fluorescent light detector (FLD) signal for MCF‐7‐, MDA‐MB‐231‐ and SK‐BR‐3‐derived EVs labelled with (B) PE‐conjugated anti‐EpCAM and (C) PE‐conjugated anti‐HER2 antibodies were plotted. (D) From FLD elution profiles, the area under the curve for the EV peak (24–80 min) was determined. Unstained EV samples were used as a negative control. (E) Different concentrations (6 × 10 9 , 8 × 10 9 , 1 × 10 10 and 2 × 10 10 particles as measured by NTA) including a negative control of LNCaP‐derived EVs (high PSMA expression) were labelled with anti‐PSMA antibodies and analysed by the AF4‐MALS‐FLD protocol. (F) The area under the curve for the EV peak was determined for LNCaP‐derived EVs. Different concentrations (2 × 10 10 , 4 × 10 10 and 6 × 10 10 particles as measured by NTA) including a negative control of (G) MCF‐7‐derived EVs (high EpCAM expression) or (I) SK‐BR‐3‐derived EVs (high HER2 expression) were labelled with PE‐conjugated anti‐EpCAM or anti‐HER2 antibodies respectively and analysed by the AF4‐MALS‐FLD protocol. The area under the curve for the EV peak (24–80 min) was determined for (H) MCF‐7‐ and (J) SK‐BR‐3‐derived EVs.

Article Snippet: The following primary and secondary antibodies were used for western blot analysis: mouse monoclonal anti‐Alix (1:1000) (cat no. 2171S, Cell Signaling Technology), rabbit monoclonal anti‐CD9 (1:1000) (cat no. 13403S, Cell Signaling Technology), rabbit monoclonal anti‐Syntenin‐1 (1:1000) (cat no. ab133267, Abcam), mouse monoclonal anti‐TSG101 (1:1000) (cat no. sc‐7964, Santa Cruz Biotechnology), rabbit monoclonal anti‐PSMA (1:1000) (cat no. 12702S), mouse monoclonal anti‐EpCAM (1:1000) (cat no. 2929S, Cell Signaling Technology), rabbit monoclonal anti‐HER2 (1:1000) (cat no. 2165S, Cell Signaling Technology), mouse monoclonal anti‐GAPDH (1:2500) (cat no. G8795, Merck Life Science), sheep anti‐mouse horseradish peroxidase‐linked (1:3000) (cat no. NA931V, GE Healthcare Life Sciences) and donkey anti‐rabbit horseradish peroxidase‐linked antibody (1:8000) (cat no. NA934V, GE Healthcare Life Sciences).

Techniques: Biomarker Discovery, Derivative Assay, Multi-Angle Light Scattering, Negative Control, Expressing

Journal: Journal of Extracellular Biology

Article Title: A One‐Step Workflow for Size‐Based Separation of Extracellular Vesicles With Integrated Surface Marker Detection

doi: 10.1002/jex2.70109

Figure Lengend Snippet: Detection of EVs in complex matrices . (A) Different volumes of cell culture supernatant (0, 20, 40 and 60 µL) collected from the MCF‐7 cells were labelled with PE‐conjugated anti‐EpCAM antibodies and analysed by AF4‐MALS‐FLD. The area under the curve for the EV peak in complex matrices (40–80 min) was determined. (B) Different amounts of LNCaP‐derived EVs were spiked in 100 µL of concentrated urine, diluted 1:1 in PBS to reduce viscosity, labelled with PE‐conjugated anti‐PSMA antibodies, and analysed by AF4‐MALS‐FLD. The area under the curve for the EV peak was determined. Different amounts of (C) MCF‐7‐ or (D) SK‐BR‐3‐derived EVs were spiked in 100 µL of blood plasma, diluted 1:1 in PBS to reduce viscosity, and labelled with PE‐conjugated anti‐EpCAM or anti‐HER2 antibodies, respectively. Labelled EVs were analysed by AF4‐MALS‐FLD and the area under the curve for the EV peak was determined. Different amounts of SK‐BR‐3 EVs were also spiked in blood plasma and labelled with isotype control antibodies. (E) Different concentrations of soluble EpCAM (1, 5 and 10 ng/mL) and soluble HER2 (50, 100 and 150 ng/mL) were spiked in blood plasma, labelled with PE‐conjugated anti‐EpCAM or anti‐HER2 antibodies respectively, and analysed by AF4‐MALS‐FLD.

Article Snippet: The following primary and secondary antibodies were used for western blot analysis: mouse monoclonal anti‐Alix (1:1000) (cat no. 2171S, Cell Signaling Technology), rabbit monoclonal anti‐CD9 (1:1000) (cat no. 13403S, Cell Signaling Technology), rabbit monoclonal anti‐Syntenin‐1 (1:1000) (cat no. ab133267, Abcam), mouse monoclonal anti‐TSG101 (1:1000) (cat no. sc‐7964, Santa Cruz Biotechnology), rabbit monoclonal anti‐PSMA (1:1000) (cat no. 12702S), mouse monoclonal anti‐EpCAM (1:1000) (cat no. 2929S, Cell Signaling Technology), rabbit monoclonal anti‐HER2 (1:1000) (cat no. 2165S, Cell Signaling Technology), mouse monoclonal anti‐GAPDH (1:2500) (cat no. G8795, Merck Life Science), sheep anti‐mouse horseradish peroxidase‐linked (1:3000) (cat no. NA931V, GE Healthcare Life Sciences) and donkey anti‐rabbit horseradish peroxidase‐linked antibody (1:8000) (cat no. NA934V, GE Healthcare Life Sciences).

Techniques: Cell Culture, Derivative Assay, Viscosity, Clinical Proteomics, Control

Journal: Journal of Extracellular Biology

Article Title: A One‐Step Workflow for Size‐Based Separation of Extracellular Vesicles With Integrated Surface Marker Detection

doi: 10.1002/jex2.70109

Figure Lengend Snippet: Validation of the AF4‐MALS‐FLD workflow on patient samples . Urine samples of five prostate cancer patients were labelled for PSMA and analysed by the AF4‐MALS‐FLD workflow. Fractions 40–80 min were collected, concentrated and processed for mass spectrometry‐based proteomic analysis. (A) EV markers Syntenin‐1, Flotillin‐1, CD63, CD9, CD81, Flotillin‐2, Alix and TSG101 were analysed (missing sample indicated in grey). Z ‐score transformation of intensities were plotted. (B) Targeted mass spectrometry analysed the presence of PSMA (FOLH1) in patient samples. The z ‐score transformation of intensities was plotted with the AF4‐MALS‐FLD peak area. (C) Blood plasma samples of healthy controls ( n = 7) and HER2 amplified breast cancer patients ( n = 10) were labelled with PE‐conjugated anti‐HER2 antibodies. (D) Blood plasma samples of healthy controls ( n = 6) and breast cancer patients ( n = 8) were labelled with PE‐conjugated anti‐EpCAM antibodies. The area under the curve values were normalised for the mean value in the healthy control group.

Article Snippet: The following primary and secondary antibodies were used for western blot analysis: mouse monoclonal anti‐Alix (1:1000) (cat no. 2171S, Cell Signaling Technology), rabbit monoclonal anti‐CD9 (1:1000) (cat no. 13403S, Cell Signaling Technology), rabbit monoclonal anti‐Syntenin‐1 (1:1000) (cat no. ab133267, Abcam), mouse monoclonal anti‐TSG101 (1:1000) (cat no. sc‐7964, Santa Cruz Biotechnology), rabbit monoclonal anti‐PSMA (1:1000) (cat no. 12702S), mouse monoclonal anti‐EpCAM (1:1000) (cat no. 2929S, Cell Signaling Technology), rabbit monoclonal anti‐HER2 (1:1000) (cat no. 2165S, Cell Signaling Technology), mouse monoclonal anti‐GAPDH (1:2500) (cat no. G8795, Merck Life Science), sheep anti‐mouse horseradish peroxidase‐linked (1:3000) (cat no. NA931V, GE Healthcare Life Sciences) and donkey anti‐rabbit horseradish peroxidase‐linked antibody (1:8000) (cat no. NA934V, GE Healthcare Life Sciences).

Techniques: Biomarker Discovery, Mass Spectrometry, Transformation Assay, Clinical Proteomics, Amplification, Control

Journal: Journal of Extracellular Biology

Article Title: A One‐Step Workflow for Size‐Based Separation of Extracellular Vesicles With Integrated Surface Marker Detection

doi: 10.1002/jex2.70109

Figure Lengend Snippet: Representation and validation of the AF4‐MALS‐FLD method . (A) Overview of the workflow used for identification of EV surface proteins. PE‐conjugated antibodies were incubated with the sample (e.g. pre‐purified EVs, cell culture supernatant, urine, or plasma) and loaded into the AF4 channel. (B) The light scatter elution profile (in relative scale) (black, full line), UV elution profile (black, dotted line) and the size determination ( R rms in nm) (red) obtained by the multi‐angle light scattering (MALS) detector is plotted against time for labelling of SK‐BR‐3‐derived EVs with PE‐conjugated anti‐CD81 antibody. (C) The fluorescent light detector (FLD) signal (in relative scale) for SK‐BR‐3‐derived EVs labelled with PE‐conjugated anti‐CD9, anti‐CD63 and anti‐CD81 is plotted against time. (D) Transmission electron microscopy (TEM) images of different fractions of the AF4‐MALS‐FLD elution profile are shown (scale bar = 200 nm).

Article Snippet: The following primary and secondary antibodies were used for western blot analysis: mouse monoclonal anti‐Alix (1:1000) (cat no. 2171S, Cell Signaling Technology), rabbit monoclonal anti‐CD9 (1:1000) (cat no. 13403S, Cell Signaling Technology), rabbit monoclonal anti‐Syntenin‐1 (1:1000) (cat no. ab133267, Abcam), mouse monoclonal anti‐TSG101 (1:1000) (cat no. sc‐7964, Santa Cruz Biotechnology), rabbit monoclonal anti‐PSMA (1:1000) (cat no. 12702S), mouse monoclonal anti‐EpCAM (1:1000) (cat no. 2929S, Cell Signaling Technology), rabbit monoclonal anti‐HER2 (1:1000) (cat no. 2165S, Cell Signaling Technology), mouse monoclonal anti‐GAPDH (1:2500) (cat no. G8795, Merck Life Science), sheep anti‐mouse horseradish peroxidase‐linked (1:3000) (cat no. NA931V, GE Healthcare Life Sciences) and donkey anti‐rabbit horseradish peroxidase‐linked antibody (1:8000) (cat no. NA934V, GE Healthcare Life Sciences).

Techniques: Biomarker Discovery, Incubation, Purification, Cell Culture, Clinical Proteomics, Multi-Angle Light Scattering, Derivative Assay, Transmission Assay, Electron Microscopy

Journal: Journal of Extracellular Biology

Article Title: A One‐Step Workflow for Size‐Based Separation of Extracellular Vesicles With Integrated Surface Marker Detection

doi: 10.1002/jex2.70109

Figure Lengend Snippet: AF4‐MALS‐FLD analysis of EV surface proteins with biomarker potential in prostate and breast cancer . MCF‐7‐, MDA‐MB‐231‐ and SK‐BR‐3‐derived EVs were labelled with PE‐conjugated anti‐EpCAM antibodies and analysed by AF4‐MALS‐FLD. (A) The elution profile (in relative scale) of the multi‐angle light scatter (MALS) detector and the size ( R rms in nm) were plotted against time. The fluorescent light detector (FLD) signal for MCF‐7‐, MDA‐MB‐231‐ and SK‐BR‐3‐derived EVs labelled with (B) PE‐conjugated anti‐EpCAM and (C) PE‐conjugated anti‐HER2 antibodies were plotted. (D) From FLD elution profiles, the area under the curve for the EV peak (24–80 min) was determined. Unstained EV samples were used as a negative control. (E) Different concentrations (6 × 10 9 , 8 × 10 9 , 1 × 10 10 and 2 × 10 10 particles as measured by NTA) including a negative control of LNCaP‐derived EVs (high PSMA expression) were labelled with anti‐PSMA antibodies and analysed by the AF4‐MALS‐FLD protocol. (F) The area under the curve for the EV peak was determined for LNCaP‐derived EVs. Different concentrations (2 × 10 10 , 4 × 10 10 and 6 × 10 10 particles as measured by NTA) including a negative control of (G) MCF‐7‐derived EVs (high EpCAM expression) or (I) SK‐BR‐3‐derived EVs (high HER2 expression) were labelled with PE‐conjugated anti‐EpCAM or anti‐HER2 antibodies respectively and analysed by the AF4‐MALS‐FLD protocol. The area under the curve for the EV peak (24–80 min) was determined for (H) MCF‐7‐ and (J) SK‐BR‐3‐derived EVs.

Article Snippet: The following primary and secondary antibodies were used for western blot analysis: mouse monoclonal anti‐Alix (1:1000) (cat no. 2171S, Cell Signaling Technology), rabbit monoclonal anti‐CD9 (1:1000) (cat no. 13403S, Cell Signaling Technology), rabbit monoclonal anti‐Syntenin‐1 (1:1000) (cat no. ab133267, Abcam), mouse monoclonal anti‐TSG101 (1:1000) (cat no. sc‐7964, Santa Cruz Biotechnology), rabbit monoclonal anti‐PSMA (1:1000) (cat no. 12702S), mouse monoclonal anti‐EpCAM (1:1000) (cat no. 2929S, Cell Signaling Technology), rabbit monoclonal anti‐HER2 (1:1000) (cat no. 2165S, Cell Signaling Technology), mouse monoclonal anti‐GAPDH (1:2500) (cat no. G8795, Merck Life Science), sheep anti‐mouse horseradish peroxidase‐linked (1:3000) (cat no. NA931V, GE Healthcare Life Sciences) and donkey anti‐rabbit horseradish peroxidase‐linked antibody (1:8000) (cat no. NA934V, GE Healthcare Life Sciences).

Techniques: Biomarker Discovery, Derivative Assay, Multi-Angle Light Scattering, Negative Control, Expressing

Journal: Journal of Extracellular Biology

Article Title: A One‐Step Workflow for Size‐Based Separation of Extracellular Vesicles With Integrated Surface Marker Detection

doi: 10.1002/jex2.70109

Figure Lengend Snippet: Detection of EVs in complex matrices . (A) Different volumes of cell culture supernatant (0, 20, 40 and 60 µL) collected from the MCF‐7 cells were labelled with PE‐conjugated anti‐EpCAM antibodies and analysed by AF4‐MALS‐FLD. The area under the curve for the EV peak in complex matrices (40–80 min) was determined. (B) Different amounts of LNCaP‐derived EVs were spiked in 100 µL of concentrated urine, diluted 1:1 in PBS to reduce viscosity, labelled with PE‐conjugated anti‐PSMA antibodies, and analysed by AF4‐MALS‐FLD. The area under the curve for the EV peak was determined. Different amounts of (C) MCF‐7‐ or (D) SK‐BR‐3‐derived EVs were spiked in 100 µL of blood plasma, diluted 1:1 in PBS to reduce viscosity, and labelled with PE‐conjugated anti‐EpCAM or anti‐HER2 antibodies, respectively. Labelled EVs were analysed by AF4‐MALS‐FLD and the area under the curve for the EV peak was determined. Different amounts of SK‐BR‐3 EVs were also spiked in blood plasma and labelled with isotype control antibodies. (E) Different concentrations of soluble EpCAM (1, 5 and 10 ng/mL) and soluble HER2 (50, 100 and 150 ng/mL) were spiked in blood plasma, labelled with PE‐conjugated anti‐EpCAM or anti‐HER2 antibodies respectively, and analysed by AF4‐MALS‐FLD.

Article Snippet: The following primary and secondary antibodies were used for western blot analysis: mouse monoclonal anti‐Alix (1:1000) (cat no. 2171S, Cell Signaling Technology), rabbit monoclonal anti‐CD9 (1:1000) (cat no. 13403S, Cell Signaling Technology), rabbit monoclonal anti‐Syntenin‐1 (1:1000) (cat no. ab133267, Abcam), mouse monoclonal anti‐TSG101 (1:1000) (cat no. sc‐7964, Santa Cruz Biotechnology), rabbit monoclonal anti‐PSMA (1:1000) (cat no. 12702S), mouse monoclonal anti‐EpCAM (1:1000) (cat no. 2929S, Cell Signaling Technology), rabbit monoclonal anti‐HER2 (1:1000) (cat no. 2165S, Cell Signaling Technology), mouse monoclonal anti‐GAPDH (1:2500) (cat no. G8795, Merck Life Science), sheep anti‐mouse horseradish peroxidase‐linked (1:3000) (cat no. NA931V, GE Healthcare Life Sciences) and donkey anti‐rabbit horseradish peroxidase‐linked antibody (1:8000) (cat no. NA934V, GE Healthcare Life Sciences).

Techniques: Cell Culture, Derivative Assay, Viscosity, Clinical Proteomics, Control

Journal: Journal of Extracellular Biology

Article Title: A One‐Step Workflow for Size‐Based Separation of Extracellular Vesicles With Integrated Surface Marker Detection

doi: 10.1002/jex2.70109

Figure Lengend Snippet: Validation of the AF4‐MALS‐FLD workflow on patient samples . Urine samples of five prostate cancer patients were labelled for PSMA and analysed by the AF4‐MALS‐FLD workflow. Fractions 40–80 min were collected, concentrated and processed for mass spectrometry‐based proteomic analysis. (A) EV markers Syntenin‐1, Flotillin‐1, CD63, CD9, CD81, Flotillin‐2, Alix and TSG101 were analysed (missing sample indicated in grey). Z ‐score transformation of intensities were plotted. (B) Targeted mass spectrometry analysed the presence of PSMA (FOLH1) in patient samples. The z ‐score transformation of intensities was plotted with the AF4‐MALS‐FLD peak area. (C) Blood plasma samples of healthy controls ( n = 7) and HER2 amplified breast cancer patients ( n = 10) were labelled with PE‐conjugated anti‐HER2 antibodies. (D) Blood plasma samples of healthy controls ( n = 6) and breast cancer patients ( n = 8) were labelled with PE‐conjugated anti‐EpCAM antibodies. The area under the curve values were normalised for the mean value in the healthy control group.

Article Snippet: The following primary and secondary antibodies were used for western blot analysis: mouse monoclonal anti‐Alix (1:1000) (cat no. 2171S, Cell Signaling Technology), rabbit monoclonal anti‐CD9 (1:1000) (cat no. 13403S, Cell Signaling Technology), rabbit monoclonal anti‐Syntenin‐1 (1:1000) (cat no. ab133267, Abcam), mouse monoclonal anti‐TSG101 (1:1000) (cat no. sc‐7964, Santa Cruz Biotechnology), rabbit monoclonal anti‐PSMA (1:1000) (cat no. 12702S), mouse monoclonal anti‐EpCAM (1:1000) (cat no. 2929S, Cell Signaling Technology), rabbit monoclonal anti‐HER2 (1:1000) (cat no. 2165S, Cell Signaling Technology), mouse monoclonal anti‐GAPDH (1:2500) (cat no. G8795, Merck Life Science), sheep anti‐mouse horseradish peroxidase‐linked (1:3000) (cat no. NA931V, GE Healthcare Life Sciences) and donkey anti‐rabbit horseradish peroxidase‐linked antibody (1:8000) (cat no. NA934V, GE Healthcare Life Sciences).

Techniques: Biomarker Discovery, Mass Spectrometry, Transformation Assay, Clinical Proteomics, Amplification, Control

Journal: Journal of Extracellular Biology

Article Title: A One‐Step Workflow for Size‐Based Separation of Extracellular Vesicles With Integrated Surface Marker Detection

doi: 10.1002/jex2.70109

Figure Lengend Snippet: Representation and validation of the AF4‐MALS‐FLD method . (A) Overview of the workflow used for identification of EV surface proteins. PE‐conjugated antibodies were incubated with the sample (e.g. pre‐purified EVs, cell culture supernatant, urine, or plasma) and loaded into the AF4 channel. (B) The light scatter elution profile (in relative scale) (black, full line), UV elution profile (black, dotted line) and the size determination ( R rms in nm) (red) obtained by the multi‐angle light scattering (MALS) detector is plotted against time for labelling of SK‐BR‐3‐derived EVs with PE‐conjugated anti‐CD81 antibody. (C) The fluorescent light detector (FLD) signal (in relative scale) for SK‐BR‐3‐derived EVs labelled with PE‐conjugated anti‐CD9, anti‐CD63 and anti‐CD81 is plotted against time. (D) Transmission electron microscopy (TEM) images of different fractions of the AF4‐MALS‐FLD elution profile are shown (scale bar = 200 nm).

Article Snippet: The following primary and secondary antibodies were used for western blot analysis: mouse monoclonal anti‐Alix (1:1000) (cat no. 2171S, Cell Signaling Technology), rabbit monoclonal anti‐CD9 (1:1000) (cat no. 13403S, Cell Signaling Technology), rabbit monoclonal anti‐Syntenin‐1 (1:1000) (cat no. ab133267, Abcam), mouse monoclonal anti‐TSG101 (1:1000) (cat no. sc‐7964, Santa Cruz Biotechnology), rabbit monoclonal anti‐PSMA (1:1000) (cat no. 12702S), mouse monoclonal anti‐EpCAM (1:1000) (cat no. 2929S, Cell Signaling Technology), rabbit monoclonal anti‐HER2 (1:1000) (cat no. 2165S, Cell Signaling Technology), mouse monoclonal anti‐GAPDH (1:2500) (cat no. G8795, Merck Life Science), sheep anti‐mouse horseradish peroxidase‐linked (1:3000) (cat no. NA931V, GE Healthcare Life Sciences) and donkey anti‐rabbit horseradish peroxidase‐linked antibody (1:8000) (cat no. NA934V, GE Healthcare Life Sciences).

Techniques: Biomarker Discovery, Incubation, Purification, Cell Culture, Clinical Proteomics, Multi-Angle Light Scattering, Derivative Assay, Transmission Assay, Electron Microscopy

Journal: Journal of Extracellular Biology

Article Title: A One‐Step Workflow for Size‐Based Separation of Extracellular Vesicles With Integrated Surface Marker Detection

doi: 10.1002/jex2.70109

Figure Lengend Snippet: AF4‐MALS‐FLD analysis of EV surface proteins with biomarker potential in prostate and breast cancer . MCF‐7‐, MDA‐MB‐231‐ and SK‐BR‐3‐derived EVs were labelled with PE‐conjugated anti‐EpCAM antibodies and analysed by AF4‐MALS‐FLD. (A) The elution profile (in relative scale) of the multi‐angle light scatter (MALS) detector and the size ( R rms in nm) were plotted against time. The fluorescent light detector (FLD) signal for MCF‐7‐, MDA‐MB‐231‐ and SK‐BR‐3‐derived EVs labelled with (B) PE‐conjugated anti‐EpCAM and (C) PE‐conjugated anti‐HER2 antibodies were plotted. (D) From FLD elution profiles, the area under the curve for the EV peak (24–80 min) was determined. Unstained EV samples were used as a negative control. (E) Different concentrations (6 × 10 9 , 8 × 10 9 , 1 × 10 10 and 2 × 10 10 particles as measured by NTA) including a negative control of LNCaP‐derived EVs (high PSMA expression) were labelled with anti‐PSMA antibodies and analysed by the AF4‐MALS‐FLD protocol. (F) The area under the curve for the EV peak was determined for LNCaP‐derived EVs. Different concentrations (2 × 10 10 , 4 × 10 10 and 6 × 10 10 particles as measured by NTA) including a negative control of (G) MCF‐7‐derived EVs (high EpCAM expression) or (I) SK‐BR‐3‐derived EVs (high HER2 expression) were labelled with PE‐conjugated anti‐EpCAM or anti‐HER2 antibodies respectively and analysed by the AF4‐MALS‐FLD protocol. The area under the curve for the EV peak (24–80 min) was determined for (H) MCF‐7‐ and (J) SK‐BR‐3‐derived EVs.

Article Snippet: The following primary and secondary antibodies were used for western blot analysis: mouse monoclonal anti‐Alix (1:1000) (cat no. 2171S, Cell Signaling Technology), rabbit monoclonal anti‐CD9 (1:1000) (cat no. 13403S, Cell Signaling Technology), rabbit monoclonal anti‐Syntenin‐1 (1:1000) (cat no. ab133267, Abcam), mouse monoclonal anti‐TSG101 (1:1000) (cat no. sc‐7964, Santa Cruz Biotechnology), rabbit monoclonal anti‐PSMA (1:1000) (cat no. 12702S), mouse monoclonal anti‐EpCAM (1:1000) (cat no. 2929S, Cell Signaling Technology), rabbit monoclonal anti‐HER2 (1:1000) (cat no. 2165S, Cell Signaling Technology), mouse monoclonal anti‐GAPDH (1:2500) (cat no. G8795, Merck Life Science), sheep anti‐mouse horseradish peroxidase‐linked (1:3000) (cat no. NA931V, GE Healthcare Life Sciences) and donkey anti‐rabbit horseradish peroxidase‐linked antibody (1:8000) (cat no. NA934V, GE Healthcare Life Sciences).

Techniques: Biomarker Discovery, Derivative Assay, Multi-Angle Light Scattering, Negative Control, Expressing

Journal: Journal of Extracellular Biology

Article Title: A One‐Step Workflow for Size‐Based Separation of Extracellular Vesicles With Integrated Surface Marker Detection

doi: 10.1002/jex2.70109

Figure Lengend Snippet: Detection of EVs in complex matrices . (A) Different volumes of cell culture supernatant (0, 20, 40 and 60 µL) collected from the MCF‐7 cells were labelled with PE‐conjugated anti‐EpCAM antibodies and analysed by AF4‐MALS‐FLD. The area under the curve for the EV peak in complex matrices (40–80 min) was determined. (B) Different amounts of LNCaP‐derived EVs were spiked in 100 µL of concentrated urine, diluted 1:1 in PBS to reduce viscosity, labelled with PE‐conjugated anti‐PSMA antibodies, and analysed by AF4‐MALS‐FLD. The area under the curve for the EV peak was determined. Different amounts of (C) MCF‐7‐ or (D) SK‐BR‐3‐derived EVs were spiked in 100 µL of blood plasma, diluted 1:1 in PBS to reduce viscosity, and labelled with PE‐conjugated anti‐EpCAM or anti‐HER2 antibodies, respectively. Labelled EVs were analysed by AF4‐MALS‐FLD and the area under the curve for the EV peak was determined. Different amounts of SK‐BR‐3 EVs were also spiked in blood plasma and labelled with isotype control antibodies. (E) Different concentrations of soluble EpCAM (1, 5 and 10 ng/mL) and soluble HER2 (50, 100 and 150 ng/mL) were spiked in blood plasma, labelled with PE‐conjugated anti‐EpCAM or anti‐HER2 antibodies respectively, and analysed by AF4‐MALS‐FLD.

Article Snippet: The following primary and secondary antibodies were used for western blot analysis: mouse monoclonal anti‐Alix (1:1000) (cat no. 2171S, Cell Signaling Technology), rabbit monoclonal anti‐CD9 (1:1000) (cat no. 13403S, Cell Signaling Technology), rabbit monoclonal anti‐Syntenin‐1 (1:1000) (cat no. ab133267, Abcam), mouse monoclonal anti‐TSG101 (1:1000) (cat no. sc‐7964, Santa Cruz Biotechnology), rabbit monoclonal anti‐PSMA (1:1000) (cat no. 12702S), mouse monoclonal anti‐EpCAM (1:1000) (cat no. 2929S, Cell Signaling Technology), rabbit monoclonal anti‐HER2 (1:1000) (cat no. 2165S, Cell Signaling Technology), mouse monoclonal anti‐GAPDH (1:2500) (cat no. G8795, Merck Life Science), sheep anti‐mouse horseradish peroxidase‐linked (1:3000) (cat no. NA931V, GE Healthcare Life Sciences) and donkey anti‐rabbit horseradish peroxidase‐linked antibody (1:8000) (cat no. NA934V, GE Healthcare Life Sciences).

Techniques: Cell Culture, Derivative Assay, Viscosity, Clinical Proteomics, Control

Journal: Journal of Extracellular Biology

Article Title: A One‐Step Workflow for Size‐Based Separation of Extracellular Vesicles With Integrated Surface Marker Detection

doi: 10.1002/jex2.70109

Figure Lengend Snippet: Validation of the AF4‐MALS‐FLD workflow on patient samples . Urine samples of five prostate cancer patients were labelled for PSMA and analysed by the AF4‐MALS‐FLD workflow. Fractions 40–80 min were collected, concentrated and processed for mass spectrometry‐based proteomic analysis. (A) EV markers Syntenin‐1, Flotillin‐1, CD63, CD9, CD81, Flotillin‐2, Alix and TSG101 were analysed (missing sample indicated in grey). Z ‐score transformation of intensities were plotted. (B) Targeted mass spectrometry analysed the presence of PSMA (FOLH1) in patient samples. The z ‐score transformation of intensities was plotted with the AF4‐MALS‐FLD peak area. (C) Blood plasma samples of healthy controls ( n = 7) and HER2 amplified breast cancer patients ( n = 10) were labelled with PE‐conjugated anti‐HER2 antibodies. (D) Blood plasma samples of healthy controls ( n = 6) and breast cancer patients ( n = 8) were labelled with PE‐conjugated anti‐EpCAM antibodies. The area under the curve values were normalised for the mean value in the healthy control group.

Article Snippet: The following primary and secondary antibodies were used for western blot analysis: mouse monoclonal anti‐Alix (1:1000) (cat no. 2171S, Cell Signaling Technology), rabbit monoclonal anti‐CD9 (1:1000) (cat no. 13403S, Cell Signaling Technology), rabbit monoclonal anti‐Syntenin‐1 (1:1000) (cat no. ab133267, Abcam), mouse monoclonal anti‐TSG101 (1:1000) (cat no. sc‐7964, Santa Cruz Biotechnology), rabbit monoclonal anti‐PSMA (1:1000) (cat no. 12702S), mouse monoclonal anti‐EpCAM (1:1000) (cat no. 2929S, Cell Signaling Technology), rabbit monoclonal anti‐HER2 (1:1000) (cat no. 2165S, Cell Signaling Technology), mouse monoclonal anti‐GAPDH (1:2500) (cat no. G8795, Merck Life Science), sheep anti‐mouse horseradish peroxidase‐linked (1:3000) (cat no. NA931V, GE Healthcare Life Sciences) and donkey anti‐rabbit horseradish peroxidase‐linked antibody (1:8000) (cat no. NA934V, GE Healthcare Life Sciences).

Techniques: Biomarker Discovery, Mass Spectrometry, Transformation Assay, Clinical Proteomics, Amplification, Control

Journal: Nucleic Acids Research

Article Title: Identification and characterization of a human MORC2 DNA binding region that is required for gene silencing

doi: 10.1093/nar/gkae1273

Figure Lengend Snippet: MORC2 preferentially associates with dsDNA via a C-terminal region. ( A ) Domain architecture of human MORC2. Structured domains are colored green. Coiled coil domains (CC), S5 transducer domain of the GHKL ATPase module (S5), a CW-type zinc finger domain (CW) and a predicted chromodomain (CD) are indicated. Purified MORC2 was cross-linked to DNA with 1 mM mechlorethamine for 30 min at 37°C or cross-linked to DNA by UV light at 254 nm for 10 min on ice, and samples were digested by trypsin. Resulting fragments were enriched by TiO 2 and analyzed by mass spectrometry (‘Materials and methods’ section). Identified amino acid positions cross-linked to DNA are denoted by a black cross. Positively charged residues and phosphorylation sites mutated in this study are noted in pink and orange, respectively, beneath the primary structure. ( B ) SDS-PAGE gel of purified human MORC2 protein (3 μg) stained with Coomassie blue stain. Contaminant band marked with an asterisk . ( C ) MORC2 DNA binding to DNA sequences of different AT/GC content as assessed by fluorescence anisotropy. Dephosphorylated MORC2 was titrated and incubated with 1 nM 5′-FAM-labeled 35 base pair duplex DNA with a high GC (71% GC content), high AT (31% GC content) or a random sequence with 49% GC content (‘Materials and methods’ section). Binding curves were fit with a single site quadratic binding equation. Error bars correspond to the standard deviation between three replicate experiments. ( D ) MORC2 binding to a 500 bp DNA substrate assessed by gel shift. Increasing concentrations of dephosphorylated MORC2 was incubated with 20 nM of duplex DNA and resolved on a 3–12% gradient Native PAGE gel (‘Materials and methods’ section). ( E ) MORC2 binding to a 1000 bp DNA substrate assessed by gel shift. Increasing concentrations of dephosphorylated MORC2 was incubated with 20 nM of duplex DNA and resolved on a 3–12% gradient Native PAGE gel (‘Materials and methods’ section). ( F ) MORC2 nucleic acid binding as measured by fluorescence anisotropy. Dephosphorylated MORC2 was titrated and incubated with 1 nM 5′-FAM-labeled 149 bp Widom 601 dsDNA, nucleosome, cruciform DNA, 35 nucleotide ssRNA and 35 base pair ssDNA (‘Materials and methods’ section). Binding curves were fit as in 1C except for the cruciform DNA which was fit with a Hill equation. Error bars correspond to the standard deviation between three replicate experiments. ( G ) MORC2 association with DNAs of varying topologies. Dephosphorylated MORC2 (150 nM) was incubated with 5′-FAM-labeled 35 base pair duplex DNA (1 nM), and positively supercoiled, negatively supercoiled, or relaxed plasmid DNA was titrated into the reactions (‘Materials and methods’ section). Binding curves were fit using an inhibition curve with a variable response. Error bars correspond to the standard deviation between three replicate experiments. ( H ) Assessment of DNA binding by dephosphorylated wild-type, aspartate mutant and 1–603 MORC2. MORC2 constructs were titrated and incubated with a FAM-labeled 35 base pair duplex DNA (1 nM) (‘Materials and methods’ section). Binding curves were fit as in 1C. Error bars correspond to the standard deviation between three replicate experiments.

Article Snippet: Beads were resuspended in 1X TBS (1 ml), with the addition of 30 μg either MORC2 antibody (Bethyl Lab #A300-149A), GFP antibody (Invitrogen #MA5-15256) or Rabbit IgG Isotype Control (Invitrogen #02–6102), and the mixture was incubated at 4°C with end-over-end mixing on a Tube Revolver (Fisher Scientific cat. no. 88861051) for 16–24 h. To immunoprecipitate MORC2, different fractions of cell lysates were incubated with Rabbit-IgG Isotype Control antibody conjugated beads at 4°C for 1 h. Lysate was clarified by centrifugation (3220 × g , 30 min, 4°C) and transferred to a new tube.

Techniques: Purification, Mass Spectrometry, Phospho-proteomics, SDS Page, Staining, Binding Assay, Fluorescence, Incubation, Labeling, Sequencing, Standard Deviation, Gel Shift, Clear Native PAGE, Plasmid Preparation, Inhibition, Mutagenesis, Construct

Journal: Nucleic Acids Research

Article Title: Identification and characterization of a human MORC2 DNA binding region that is required for gene silencing

doi: 10.1093/nar/gkae1273

Figure Lengend Snippet: Guide RNA sequences used to generate MORC2 knockout cells

Article Snippet: Beads were resuspended in 1X TBS (1 ml), with the addition of 30 μg either MORC2 antibody (Bethyl Lab #A300-149A), GFP antibody (Invitrogen #MA5-15256) or Rabbit IgG Isotype Control (Invitrogen #02–6102), and the mixture was incubated at 4°C with end-over-end mixing on a Tube Revolver (Fisher Scientific cat. no. 88861051) for 16–24 h. To immunoprecipitate MORC2, different fractions of cell lysates were incubated with Rabbit-IgG Isotype Control antibody conjugated beads at 4°C for 1 h. Lysate was clarified by centrifugation (3220 × g , 30 min, 4°C) and transferred to a new tube.

Techniques: Knock-Out

Journal: Nucleic Acids Research

Article Title: Identification and characterization of a human MORC2 DNA binding region that is required for gene silencing

doi: 10.1093/nar/gkae1273

Figure Lengend Snippet: PCR primers used to verify MORC2 knockout cells

Article Snippet: Beads were resuspended in 1X TBS (1 ml), with the addition of 30 μg either MORC2 antibody (Bethyl Lab #A300-149A), GFP antibody (Invitrogen #MA5-15256) or Rabbit IgG Isotype Control (Invitrogen #02–6102), and the mixture was incubated at 4°C with end-over-end mixing on a Tube Revolver (Fisher Scientific cat. no. 88861051) for 16–24 h. To immunoprecipitate MORC2, different fractions of cell lysates were incubated with Rabbit-IgG Isotype Control antibody conjugated beads at 4°C for 1 h. Lysate was clarified by centrifugation (3220 × g , 30 min, 4°C) and transferred to a new tube.

Techniques: Knock-Out

Journal: Nucleic Acids Research

Article Title: Identification and characterization of a human MORC2 DNA binding region that is required for gene silencing

doi: 10.1093/nar/gkae1273

Figure Lengend Snippet: MORC2 phosphorylation influences DNA binding and nuclear localization. ( A ) Phosphorylation reduces MORC2 affinity for DNA. Dephosphorylated, phosphorylated, phosphodead and phosphomimetic MORC2 were assessed for DNA binding using fluorescence anisotropy. MORC2 was titrated with FAM-labeled 35 base pair duplex DNA (1 nM) (‘Materials and methods’ section). Binding curves were fit using a quadratic binding equation. Error bars correspond to the standard deviation between three replicate experiments. ( B ) Effect of phosphorylation mutations on MORC2 localization in interphase HeLa cells. Representative confocal microscopy images of interphase HeLa cells overexpressing EGFP-wild-type MORC2, EGFP-MORC2 1–603, EGFP- MORC2 734–771, EGFP- phosphodead MORC2 and EGFP- phosphomimetic MORC2.

Article Snippet: Beads were resuspended in 1X TBS (1 ml), with the addition of 30 μg either MORC2 antibody (Bethyl Lab #A300-149A), GFP antibody (Invitrogen #MA5-15256) or Rabbit IgG Isotype Control (Invitrogen #02–6102), and the mixture was incubated at 4°C with end-over-end mixing on a Tube Revolver (Fisher Scientific cat. no. 88861051) for 16–24 h. To immunoprecipitate MORC2, different fractions of cell lysates were incubated with Rabbit-IgG Isotype Control antibody conjugated beads at 4°C for 1 h. Lysate was clarified by centrifugation (3220 × g , 30 min, 4°C) and transferred to a new tube.

Techniques: Phospho-proteomics, Binding Assay, Fluorescence, Labeling, Standard Deviation, Confocal Microscopy

Journal: Nucleic Acids Research

Article Title: Identification and characterization of a human MORC2 DNA binding region that is required for gene silencing

doi: 10.1093/nar/gkae1273

Figure Lengend Snippet: DNA binding by MORC2 reduces ATPase activity. ( A ) Assessment of MORC2 ATPase activity. Dephosphorylated wild-type, 1–603, DNA binding deficient aspartate mutant, and ATP hydrolysis deficient mutant E35A, and ATP binding deficient mutant N39A MORC2 (1 μM) were incubated with 1 mM ATP for 45 min at 37°C either in the presence or absence of a 35 base pair DNA (2 μM). Inorganic phosphate released was quantified by malachite green (‘Materials and methods’ section). Error bars correspond to the standard deviation between three replicate experiments. ( B ) MORC2 ATPase activity is reduced in the presence of DNA. Dephosphorylated MORC2 (1 μM) was incubated with 1 mM ATP for 45 min at 37°C with a titration of a 35 base pair duplex DNA. Inorganic phosphate released was quantified by malachite green (‘Materials and methods’ section). Data were fit to an inhibition curve. Error bars correspond to the standard deviation between three replicate experiments. ( C ) Michaelis–Menten analysis of MORC2 ATPase activity in the presence of DNA. Dephosphorylated MORC2 (1 μM) was incubated with an ATP titration for 45 min at 37°C in the presence of various concentrations of 35mer DNA (0–1 μM). Inorganic phosphate released was quantified by malachite green (‘Materials and methods’ section). Data were fit to a Michaelis–Menten model of enzyme kinetics. Error bars correspond to the standard deviation between three replicate experiments.

Article Snippet: Beads were resuspended in 1X TBS (1 ml), with the addition of 30 μg either MORC2 antibody (Bethyl Lab #A300-149A), GFP antibody (Invitrogen #MA5-15256) or Rabbit IgG Isotype Control (Invitrogen #02–6102), and the mixture was incubated at 4°C with end-over-end mixing on a Tube Revolver (Fisher Scientific cat. no. 88861051) for 16–24 h. To immunoprecipitate MORC2, different fractions of cell lysates were incubated with Rabbit-IgG Isotype Control antibody conjugated beads at 4°C for 1 h. Lysate was clarified by centrifugation (3220 × g , 30 min, 4°C) and transferred to a new tube.

Techniques: Binding Assay, Activity Assay, Mutagenesis, Incubation, Standard Deviation, Titration, Inhibition

Journal: Nucleic Acids Research

Article Title: Identification and characterization of a human MORC2 DNA binding region that is required for gene silencing

doi: 10.1093/nar/gkae1273

Figure Lengend Snippet: MORC2 homodimerizes via two distinct interfaces. ( A ) Dephosphorylated full-length MORC2 dimerizes in the presence and absence of AMP-PNP. Size-exclusion chromatography coupled to multi-angle light scattering (SEC-MALS) experiments with wild-type (monomer MW = 117 kDa) and 1–603 (monomer MW = 70 kDa) MORC2 in the presence or absence of 1 mM AMP-PNP. 2 mg/ml of MORC2 was applied to a WC-030 column (Wyatt Technology), and elution was monitored by absorption at 280 nm (left y -axis). MALS analysis of molecular weight is shown on the right y -axis, and average molecular weight calculations are shown across the center of each peak. ( B ) AlphaFold Multimer model of coiled coil 3 domain dimer. Chain A is colored gray and chain B is colored by pLDDT score. ( C ) MORC2 coiled coil 3 dimerizes in solution. SEC-MALS experiment performed with 1.5 mg/ml of purified MORC2 coiled coil 3 (monomer MW = 16 kDa) on a WC-010 column (Wyatt Technology). Elution was monitored by absorption at 280 nm (left y -axis). MALS analysis of molecular weight is shown on the right y -axis, and the average molecular calculation is shown across the center of the peak.

Article Snippet: Beads were resuspended in 1X TBS (1 ml), with the addition of 30 μg either MORC2 antibody (Bethyl Lab #A300-149A), GFP antibody (Invitrogen #MA5-15256) or Rabbit IgG Isotype Control (Invitrogen #02–6102), and the mixture was incubated at 4°C with end-over-end mixing on a Tube Revolver (Fisher Scientific cat. no. 88861051) for 16–24 h. To immunoprecipitate MORC2, different fractions of cell lysates were incubated with Rabbit-IgG Isotype Control antibody conjugated beads at 4°C for 1 h. Lysate was clarified by centrifugation (3220 × g , 30 min, 4°C) and transferred to a new tube.

Techniques: Size-exclusion Chromatography, Multi-Angle Light Scattering, Molecular Weight, Purification

Journal: Nucleic Acids Research

Article Title: Identification and characterization of a human MORC2 DNA binding region that is required for gene silencing

doi: 10.1093/nar/gkae1273

Figure Lengend Snippet: MORC2 can capture circular DNA substrates. ( A ) Schematic of assay to assess MORC2 capture of linear or circular DNA substrates. ( B ) Dephosphorylated, N-terminally-MBP-tagged wild-type, aspartate mutant and E35A MORC2 (600 nM) were incubated with supercoiled or linear pUC19 (100 nM). Samples were then added to amylose resin and washed in either low salt (50 mM NaCl) or high salt (400 mM NaCl) containing buffers before eluting the samples from the beads with maltose. Eluted samples were treated with proteinase K. DNA was resolved on a 1% (w/v) TAE agarose gel. Quantification of the band intensity from input and retained DNA bands normalized to the background are shown below each gel. Error bars correspond to the standard deviation between three replicate experiments. ( C ) Schematic of assay to assess MORC2 association with two circular DNA substrates. ( D ) A biotin-tagged DNA was conjugated to streptavidin magnetic beads to create a pseudo circular substrate. Dephosphorylated MORC2 (600 nM) was incubated with 20 μl of the beads in the presence or absence of 1 mM AMP-PNP. Supercoiled pBlueScript plasmid DNA (200 nM) was added, and 1 mM AMP-PNP was added or omitted before washing the beads with either low salt (50 mM NaCl) or high salt (400 mM NaCl) containing buffer. Samples were resuspended in 1X CutSmart buffer (New England Biolabs). DNA was released from the beads by digestion with ScaI and SbfI at 37°C for 1 h before proteinase K treatment. DNA was resolved on a 1% (w/v) TAE agarose gel. The intensity of the first DNA and second DNA substrate bands were quantified, normalized to the background and are presented as a ratio of second DNA:first DNA band intensity. Error bars correspond to the standard deviation between three replicate experiments. ( E ) Schematic of assay to assess MORC2 association with three circular DNA substrates. A biotin-tagged DNA was conjugated to streptavidin magnetic beads to create a pseudo-circular substrate. Dephosphorylated MORC2 (600 nM) was incubated with 20 μl of the beads. Supercoiled pBlueScript plasmid DNA (100 nM) and pBlueScript-601 plasmid DNA (100 nM) were added and 1 mM AMP-PNP was added or omitted before washing the beads with either low salt (50 mM NaCl) or high salt (400 mM NaCl) containing buffer. Samples were resuspended in 1X CutSmart buffer (New England Biolabs). DNA was released from the beads by digestion with ScaI and SbfI at 37°C for 1 h before proteinase K treatment. DNA was resolved on a 1% (w/v) TAE agarose gel.

Article Snippet: Beads were resuspended in 1X TBS (1 ml), with the addition of 30 μg either MORC2 antibody (Bethyl Lab #A300-149A), GFP antibody (Invitrogen #MA5-15256) or Rabbit IgG Isotype Control (Invitrogen #02–6102), and the mixture was incubated at 4°C with end-over-end mixing on a Tube Revolver (Fisher Scientific cat. no. 88861051) for 16–24 h. To immunoprecipitate MORC2, different fractions of cell lysates were incubated with Rabbit-IgG Isotype Control antibody conjugated beads at 4°C for 1 h. Lysate was clarified by centrifugation (3220 × g , 30 min, 4°C) and transferred to a new tube.

Techniques: Mutagenesis, Incubation, Agarose Gel Electrophoresis, Standard Deviation, Magnetic Beads, Plasmid Preparation

Journal: Nucleic Acids Research

Article Title: Identification and characterization of a human MORC2 DNA binding region that is required for gene silencing

doi: 10.1093/nar/gkae1273

Figure Lengend Snippet: DNA binding regulates MORC2 gene silencing activity in cells. ( A ) Schematic of RNAseq experiment. MORC2 constructs were added to HeLa cells lacking MORC2. MORC2 expression was induced for 48 h with doxycycline after which RNAs were extracted and sequenced. Volcano plots of RNAseq reads for overexpression of wild-type MORC2 versus control, overexpression of aspartate mutant MORC2 versus control, and wild-type MORC2 versus aspartate mutant MORC2 with spike normalization (‘Materials and methods’ section). Significant upregulated genes are shown in green, and significant downregulated genes are shown in purple from three biological replicates. Significant genes are classified as those that meet the fold change > 1.5 and FDR > 0.05 cutoffs. ( B ) Fold repression (-log 2 fold change) analysis of intronless and long-exon containing genes exhibiting a significant degree of repression after overexpression of wild-type MORC2 ( n = 75) in comparison to their fold repression after overexpression of aspartate mutant MORC2. Error bars correspond to the minimum and maximum values and the central bar represents the median value. Dotted lines connect genes between the two conditions. ( C ) Average fold repression (-log 2 fold change) of retrotransposon subfamilies as measured by RNAseq in cells with overexpression of wild-type or aspartate mutant MORC2 versus control cells using the TEtranscripts tool. Error bars represent the standard error between the elements in each subfamily (L1Hs n = 1, L1PA n = 30, L1M n = 86 and LTR-retrotransposons n = 347). ( D ) Model of how MORC2 engages DNA to promote compaction. MORC2 contains a DNA binding region between the GHKL domain and coiled coil 3 domain dimerization interfaces. DNA binding inside the lumen of the dimer communicates to the ATPase domain, to favor an ATP-bound homodimer conformation that is incompatible with ATP hydrolysis. MORC2 association with multiple DNA segments may allow MORC2 to bridge distal regions of DNA to contribute to compaction.

Article Snippet: Beads were resuspended in 1X TBS (1 ml), with the addition of 30 μg either MORC2 antibody (Bethyl Lab #A300-149A), GFP antibody (Invitrogen #MA5-15256) or Rabbit IgG Isotype Control (Invitrogen #02–6102), and the mixture was incubated at 4°C with end-over-end mixing on a Tube Revolver (Fisher Scientific cat. no. 88861051) for 16–24 h. To immunoprecipitate MORC2, different fractions of cell lysates were incubated with Rabbit-IgG Isotype Control antibody conjugated beads at 4°C for 1 h. Lysate was clarified by centrifugation (3220 × g , 30 min, 4°C) and transferred to a new tube.

Techniques: Binding Assay, Activity Assay, Construct, Expressing, Over Expression, Control, Mutagenesis, Comparison

Journal: Journal of Extracellular Biology

Article Title: A One‐Step Workflow for Size‐Based Separation of Extracellular Vesicles With Integrated Surface Marker Detection

doi: 10.1002/jex2.70109

Figure Lengend Snippet: Representation and validation of the AF4‐MALS‐FLD method . (A) Overview of the workflow used for identification of EV surface proteins. PE‐conjugated antibodies were incubated with the sample (e.g. pre‐purified EVs, cell culture supernatant, urine, or plasma) and loaded into the AF4 channel. (B) The light scatter elution profile (in relative scale) (black, full line), UV elution profile (black, dotted line) and the size determination ( R rms in nm) (red) obtained by the multi‐angle light scattering (MALS) detector is plotted against time for labelling of SK‐BR‐3‐derived EVs with PE‐conjugated anti‐CD81 antibody. (C) The fluorescent light detector (FLD) signal (in relative scale) for SK‐BR‐3‐derived EVs labelled with PE‐conjugated anti‐CD9, anti‐CD63 and anti‐CD81 is plotted against time. (D) Transmission electron microscopy (TEM) images of different fractions of the AF4‐MALS‐FLD elution profile are shown (scale bar = 200 nm).

Article Snippet: The following primary and secondary antibodies were used for western blot analysis: mouse monoclonal anti‐Alix (1:1000) (cat no. 2171S, Cell Signaling Technology), rabbit monoclonal anti‐CD9 (1:1000) (cat no. 13403S, Cell Signaling Technology), rabbit monoclonal anti‐Syntenin‐1 (1:1000) (cat no. ab133267, Abcam), mouse monoclonal anti‐TSG101 (1:1000) (cat no. sc‐7964, Santa Cruz Biotechnology), rabbit monoclonal anti‐PSMA (1:1000) (cat no. 12702S), mouse monoclonal anti‐EpCAM (1:1000) (cat no. 2929S, Cell Signaling Technology), rabbit monoclonal anti‐HER2 (1:1000) (cat no. 2165S, Cell Signaling Technology), mouse monoclonal anti‐GAPDH (1:2500) (cat no. G8795, Merck Life Science), sheep anti‐mouse horseradish peroxidase‐linked (1:3000) (cat no. NA931V, GE Healthcare Life Sciences) and donkey anti‐rabbit horseradish peroxidase‐linked antibody (1:8000) (cat no. NA934V, GE Healthcare Life Sciences).

Techniques: Biomarker Discovery, Incubation, Purification, Cell Culture, Clinical Proteomics, Multi-Angle Light Scattering, Derivative Assay, Transmission Assay, Electron Microscopy

Journal: Journal of Extracellular Biology

Article Title: A One‐Step Workflow for Size‐Based Separation of Extracellular Vesicles With Integrated Surface Marker Detection

doi: 10.1002/jex2.70109

Figure Lengend Snippet: AF4‐MALS‐FLD analysis of EV surface proteins with biomarker potential in prostate and breast cancer . MCF‐7‐, MDA‐MB‐231‐ and SK‐BR‐3‐derived EVs were labelled with PE‐conjugated anti‐EpCAM antibodies and analysed by AF4‐MALS‐FLD. (A) The elution profile (in relative scale) of the multi‐angle light scatter (MALS) detector and the size ( R rms in nm) were plotted against time. The fluorescent light detector (FLD) signal for MCF‐7‐, MDA‐MB‐231‐ and SK‐BR‐3‐derived EVs labelled with (B) PE‐conjugated anti‐EpCAM and (C) PE‐conjugated anti‐HER2 antibodies were plotted. (D) From FLD elution profiles, the area under the curve for the EV peak (24–80 min) was determined. Unstained EV samples were used as a negative control. (E) Different concentrations (6 × 10 9 , 8 × 10 9 , 1 × 10 10 and 2 × 10 10 particles as measured by NTA) including a negative control of LNCaP‐derived EVs (high PSMA expression) were labelled with anti‐PSMA antibodies and analysed by the AF4‐MALS‐FLD protocol. (F) The area under the curve for the EV peak was determined for LNCaP‐derived EVs. Different concentrations (2 × 10 10 , 4 × 10 10 and 6 × 10 10 particles as measured by NTA) including a negative control of (G) MCF‐7‐derived EVs (high EpCAM expression) or (I) SK‐BR‐3‐derived EVs (high HER2 expression) were labelled with PE‐conjugated anti‐EpCAM or anti‐HER2 antibodies respectively and analysed by the AF4‐MALS‐FLD protocol. The area under the curve for the EV peak (24–80 min) was determined for (H) MCF‐7‐ and (J) SK‐BR‐3‐derived EVs.

Article Snippet: The following primary and secondary antibodies were used for western blot analysis: mouse monoclonal anti‐Alix (1:1000) (cat no. 2171S, Cell Signaling Technology), rabbit monoclonal anti‐CD9 (1:1000) (cat no. 13403S, Cell Signaling Technology), rabbit monoclonal anti‐Syntenin‐1 (1:1000) (cat no. ab133267, Abcam), mouse monoclonal anti‐TSG101 (1:1000) (cat no. sc‐7964, Santa Cruz Biotechnology), rabbit monoclonal anti‐PSMA (1:1000) (cat no. 12702S), mouse monoclonal anti‐EpCAM (1:1000) (cat no. 2929S, Cell Signaling Technology), rabbit monoclonal anti‐HER2 (1:1000) (cat no. 2165S, Cell Signaling Technology), mouse monoclonal anti‐GAPDH (1:2500) (cat no. G8795, Merck Life Science), sheep anti‐mouse horseradish peroxidase‐linked (1:3000) (cat no. NA931V, GE Healthcare Life Sciences) and donkey anti‐rabbit horseradish peroxidase‐linked antibody (1:8000) (cat no. NA934V, GE Healthcare Life Sciences).

Techniques: Biomarker Discovery, Derivative Assay, Multi-Angle Light Scattering, Negative Control, Expressing

Journal: Journal of Extracellular Biology

Article Title: A One‐Step Workflow for Size‐Based Separation of Extracellular Vesicles With Integrated Surface Marker Detection

doi: 10.1002/jex2.70109

Figure Lengend Snippet: Detection of EVs in complex matrices . (A) Different volumes of cell culture supernatant (0, 20, 40 and 60 µL) collected from the MCF‐7 cells were labelled with PE‐conjugated anti‐EpCAM antibodies and analysed by AF4‐MALS‐FLD. The area under the curve for the EV peak in complex matrices (40–80 min) was determined. (B) Different amounts of LNCaP‐derived EVs were spiked in 100 µL of concentrated urine, diluted 1:1 in PBS to reduce viscosity, labelled with PE‐conjugated anti‐PSMA antibodies, and analysed by AF4‐MALS‐FLD. The area under the curve for the EV peak was determined. Different amounts of (C) MCF‐7‐ or (D) SK‐BR‐3‐derived EVs were spiked in 100 µL of blood plasma, diluted 1:1 in PBS to reduce viscosity, and labelled with PE‐conjugated anti‐EpCAM or anti‐HER2 antibodies, respectively. Labelled EVs were analysed by AF4‐MALS‐FLD and the area under the curve for the EV peak was determined. Different amounts of SK‐BR‐3 EVs were also spiked in blood plasma and labelled with isotype control antibodies. (E) Different concentrations of soluble EpCAM (1, 5 and 10 ng/mL) and soluble HER2 (50, 100 and 150 ng/mL) were spiked in blood plasma, labelled with PE‐conjugated anti‐EpCAM or anti‐HER2 antibodies respectively, and analysed by AF4‐MALS‐FLD.

Article Snippet: The following primary and secondary antibodies were used for western blot analysis: mouse monoclonal anti‐Alix (1:1000) (cat no. 2171S, Cell Signaling Technology), rabbit monoclonal anti‐CD9 (1:1000) (cat no. 13403S, Cell Signaling Technology), rabbit monoclonal anti‐Syntenin‐1 (1:1000) (cat no. ab133267, Abcam), mouse monoclonal anti‐TSG101 (1:1000) (cat no. sc‐7964, Santa Cruz Biotechnology), rabbit monoclonal anti‐PSMA (1:1000) (cat no. 12702S), mouse monoclonal anti‐EpCAM (1:1000) (cat no. 2929S, Cell Signaling Technology), rabbit monoclonal anti‐HER2 (1:1000) (cat no. 2165S, Cell Signaling Technology), mouse monoclonal anti‐GAPDH (1:2500) (cat no. G8795, Merck Life Science), sheep anti‐mouse horseradish peroxidase‐linked (1:3000) (cat no. NA931V, GE Healthcare Life Sciences) and donkey anti‐rabbit horseradish peroxidase‐linked antibody (1:8000) (cat no. NA934V, GE Healthcare Life Sciences).

Techniques: Cell Culture, Derivative Assay, Viscosity, Clinical Proteomics, Control

Journal: Journal of Extracellular Biology

Article Title: A One‐Step Workflow for Size‐Based Separation of Extracellular Vesicles With Integrated Surface Marker Detection

doi: 10.1002/jex2.70109

Figure Lengend Snippet: Validation of the AF4‐MALS‐FLD workflow on patient samples . Urine samples of five prostate cancer patients were labelled for PSMA and analysed by the AF4‐MALS‐FLD workflow. Fractions 40–80 min were collected, concentrated and processed for mass spectrometry‐based proteomic analysis. (A) EV markers Syntenin‐1, Flotillin‐1, CD63, CD9, CD81, Flotillin‐2, Alix and TSG101 were analysed (missing sample indicated in grey). Z ‐score transformation of intensities were plotted. (B) Targeted mass spectrometry analysed the presence of PSMA (FOLH1) in patient samples. The z ‐score transformation of intensities was plotted with the AF4‐MALS‐FLD peak area. (C) Blood plasma samples of healthy controls ( n = 7) and HER2 amplified breast cancer patients ( n = 10) were labelled with PE‐conjugated anti‐HER2 antibodies. (D) Blood plasma samples of healthy controls ( n = 6) and breast cancer patients ( n = 8) were labelled with PE‐conjugated anti‐EpCAM antibodies. The area under the curve values were normalised for the mean value in the healthy control group.

Article Snippet: The following primary and secondary antibodies were used for western blot analysis: mouse monoclonal anti‐Alix (1:1000) (cat no. 2171S, Cell Signaling Technology), rabbit monoclonal anti‐CD9 (1:1000) (cat no. 13403S, Cell Signaling Technology), rabbit monoclonal anti‐Syntenin‐1 (1:1000) (cat no. ab133267, Abcam), mouse monoclonal anti‐TSG101 (1:1000) (cat no. sc‐7964, Santa Cruz Biotechnology), rabbit monoclonal anti‐PSMA (1:1000) (cat no. 12702S), mouse monoclonal anti‐EpCAM (1:1000) (cat no. 2929S, Cell Signaling Technology), rabbit monoclonal anti‐HER2 (1:1000) (cat no. 2165S, Cell Signaling Technology), mouse monoclonal anti‐GAPDH (1:2500) (cat no. G8795, Merck Life Science), sheep anti‐mouse horseradish peroxidase‐linked (1:3000) (cat no. NA931V, GE Healthcare Life Sciences) and donkey anti‐rabbit horseradish peroxidase‐linked antibody (1:8000) (cat no. NA934V, GE Healthcare Life Sciences).

Techniques: Biomarker Discovery, Mass Spectrometry, Transformation Assay, Clinical Proteomics, Amplification, Control