Journal: Cancer research

Article Title: Mutant IDH1 Cooperates with ATRX Loss to Drive the Alternative Lengthening of Telomere Phenotype in Glioma

doi: 10.1158/0008-5472.CAN-17-2269

Figure Lengend Snippet: Mutant IDH1 expression is associated with downregulation of RAP1 and XRCC1. A, Triplicate quantitative PCR analysis of the levels of select transcripts encoding proteins involved in telomere regulation in control cells (E6E7 astrocytes) expressing mutant IDH1, subjected to CRISPR-based deletion of ATRX, or both. B, Western blot analysis of RAP1, XRCC1, DNA ligase 3 (Lig3), and β-actin protein levels in the cells in A and in BT142, SF10602, and MGG119 IDH1-mutant ALT glioma cells. C, The Cancer Genome Atlas–based analysis of XRCC1 and RAP1 mRNA levels in LGA, lower-grade mixed oligoastroglioma (LOA), and GBM relative to normal brain controls (1.0 on the y-axis). *, P < 0.05.

Article Snippet: SF10602 cells were provided by the UCSF Brain Tumor Center Tissue Core, BT142 cells were purchased from ATCC, GM847 were purchased from The Coriell Institute (Camden, NJ), and MGG119 human glioma cells were a gift from Daniel Cahill (Massachusetts General, Boston, MA).

Techniques: Mutagenesis, Expressing, Real-time Polymerase Chain Reaction, Control, CRISPR, Western Blot

Journal: Redox Biology

Article Title: Oxidative damage to lung mitochondrial DNA is a key contributor to the development of chemical lung injury

doi: 10.1016/j.redox.2025.103624

Figure Lengend Snippet: Adult male and female (C57BL/6) mice were exposed to Cl 2 gas (500 ppm for 30 min) and returned to room air. One hour later, mitoOGG1 (1 mg/kg BW in 50 μl buffer) or vehicle were instilled intranasally. Mice were sacrificed and lung tissues were collected at different time points post Cl 2 exposure. ( A ) mRNA expression of OGG1 was measured by real-time PCR. Data are shown as fold change after normalization with the housekeeping gene (18s); means ± 1 SEM, each symbol represents data from a different mouse. ( B ) Immunoblot of lung tissues using antibodies against mouse OGG1 and β-tubulin at the indicated conditions. Each lane represents data from a different mouse. ( C ) Fold changes of lung OGG1 proteins of data shown in B at 24 h post exposure. Data are normalized to the corresponding values of β-tubulin. Each symbol represents data from a different mouse. ( D ) PCR Amplification of long (∼11 kb) and short fragments (∼100bp) of mitochondrial DNA (mtDNA) using genomic DNA isolated from lung tissues from air, post 24hrs of Cl 2 and following instillation of vehicle or mitoOGG1 (1 mg/kg BW in 50 μl of buffer) at 1 h post exposure. ( E ) Intact mtDNA in lung tissue (expressed as fold change of the air value), assessed by RT-PCR for the indicated conditions; means ± 1 SEM, each symbol represents data from a different mouse. ( F) Intact lung mtDNA injury at 1 h post exposure measured by Southern blots in the mitochondria D-loop in the presence and absence of Fpg; means ± 1 SEM, each symbol represents data from a different mouse. ( G ) Immunoblot of lung tissues with antibodies against GAPDH (a cytoplasmic marker) and Tom20, an outer mitochondrial membrane protein. Each lane represents data from a different mouse. ( H ) Dot blot showing 8-Oxo-dG DNA lesions in mtDNA of lung tissue for the indicated conditions. Each dot represents a single animal. ( I ). Fixed tissues from the right lung, upper lobe of mice at the indicated conditions (Air, post 24hrs of Cl 2 and Cl 2 with mitoOGG1),immunostained with antibodies against 8-oxo-dG (Green) and Tom20 (Red). Colocalization of 8-oxo-dG and Tom20 (orange color) indicates the presence of 8-Oxo-dG in the mitochondria at 24 h post Cl 2 . One slide per mouse; n = 3 mice for each group. ( J ) Transmission electron microscopy images of mitochondria with abnormal morphology in mice lungs. Damaged mitochondria with matrix swelling and collapsed cristae was observed post Cl 2 exposure. Scale bar; 1 μm, 600 nm. Data from three slides for one mouse for each group. Data are shown in fold change and SEM; One-way ANOVA followed by the Tukey t -test adjusted for multiple comparisons (PRISM 10).

Article Snippet: The membrane was incubated with OGG1 (Cat No. 15125-1-AP, Proteintech), GAPDH (Cat No. 60004-1-Ig, Proteintech), and β-Tubulin (Cat No. 10094-1-AP, Proteintech), β-Actin (Cat No. 66009-1-Ig, Proteintech), 6∗His-Tag (Cat No. 66005-1-Ig, Proteintech), OGG1 (NB100-106, Novus) in 5 % BSA, 1xTBS, 0.1 % Tween 20 at 4 °C with gentle shaking, for overnight at 4 °C followed by incubation with HRP-conjugated secondary antibodies against mouse (Cat No. 1721011, Bio- Rad Hercules, CA) and rabbit (Cat No. 1706515, Bio-Rad, Hercules, CA) at room temperature for 1 h. Then, images were captured on Syngene Imager by using Supersignal West Dura Extended duration substrate (Cat No. 34076, Thermo scientific).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Amplification, Isolation, Reverse Transcription Polymerase Chain Reaction, Marker, Membrane, Dot Blot, Transmission Assay, Electron Microscopy

Journal: Redox Biology

Article Title: Oxidative damage to lung mitochondrial DNA is a key contributor to the development of chemical lung injury

doi: 10.1016/j.redox.2025.103624

Figure Lengend Snippet: Distribution of intranasally instilled 89 Z r -mitoOGG1 in air and Cl 2 exposed mice . Male and female C57BL/6 mice were exposed to air or Cl 2 (500 ppm for 30min) and returned to room air. Three hours later, 89 Zr-mitoOGG1 (1 mg/kg BW in 50 μl of buffer) was instilled intranasally. ( A ) Radio-iTLC of 89 Zr-OGG1 (R f of 89 Zr-mitoOGG1 is 0, R f free 89 Zr ∼ 1). ( B ) Mean Standard Uptake Values (SUV mean ) ±1 SEM (n = 4 mice per group) of 89 Zr-mitoOGG1 levels measured in the lungs of mice in the control (post air) and treated (post Cl 2 gas exposed) mice over time ( C ) SUV mean comparison of heart, liver, kidney, muscle and lungs at various points post instillation in the Cl 2 exposed mice. SUV mean values are means ± 1 SEM; each symbol represents an individual mouse. (D) Maximum Intensity Projection (MIP) and axial slice PET/CT images of intranasally instilled 89 Zr-mitoOGG1 at 6 h and 24 h post-instillation in control/air and Cl 2 exposed mice. (E) Biodistribution of 89 Zr-mitoOGG1 in control/air and Cl 2 exposed mice at 24 h post instillation. n = 4 for each group. Data are expressed at % injected dose/gram (%ID/g), avg = average and SD = standard deviation.

Article Snippet: The membrane was incubated with OGG1 (Cat No. 15125-1-AP, Proteintech), GAPDH (Cat No. 60004-1-Ig, Proteintech), and β-Tubulin (Cat No. 10094-1-AP, Proteintech), β-Actin (Cat No. 66009-1-Ig, Proteintech), 6∗His-Tag (Cat No. 66005-1-Ig, Proteintech), OGG1 (NB100-106, Novus) in 5 % BSA, 1xTBS, 0.1 % Tween 20 at 4 °C with gentle shaking, for overnight at 4 °C followed by incubation with HRP-conjugated secondary antibodies against mouse (Cat No. 1721011, Bio- Rad Hercules, CA) and rabbit (Cat No. 1706515, Bio-Rad, Hercules, CA) at room temperature for 1 h. Then, images were captured on Syngene Imager by using Supersignal West Dura Extended duration substrate (Cat No. 34076, Thermo scientific).

Techniques: Control, Comparison, Positron Emission Tomography-Computed Tomography, Injection, Standard Deviation

Journal: Redox Biology

Article Title: Oxidative damage to lung mitochondrial DNA is a key contributor to the development of chemical lung injury

doi: 10.1016/j.redox.2025.103624

Figure Lengend Snippet: Instillation of mitoOGG1 improved survival and decreased lung injury at 14 d post exposure . Male and Female C57BL/6 mice were exposed to air or Cl 2 (400 ppm for 30 min) and returned to room air. Post 6 h of Cl 2 exposure, mice were intranasally instilled with mitoOGG1 (0.5 mg/kg BW in 50 μl of sterile saline) and saline and were kept under observation for 21 days ( A) Kaplan-Meier curve showing improved survival in intranasally instilled mitoOGG1 mice as compared to the saline group; statistical significance was calculated by using GraphPad PRISM. ( B ) Quasi-static pressure volume loops measured by flexiVent at 14 days post exposure of male and female C57 mice to air or Cl 2 . Inflation and deflation curves are shown for each group. Lung compliance is measured from the linear portion of the deflation loop. Means ± 1 SEM; n = 9 for air; n = 5 for Cl 2 and n = 4 for Cl 2 +mitoOGG1. ( C ) Total lung capacity (TLC) defined as the lung volume at 22 cmH2O pressure for the indicated groups. Values are means ± SEM; n = 4–6 for each group. 1-way ANOVA with Tukey post-test. ( D ) Transgenic mice with global (mitochondrial and nuclear) OGG1 depletion (OGG1 −/− ) exposed to Cl 2 exhibited much higher mortality as compared to their littermate controls in the FBV and C57B/L6 background. Kaplan-Meyer Curves generated by PRISM. Statistical Analysis by the Log-rank (Mantel-Cox) test (PRISM). Numbers indicate the number of mice for Wild-type controls (n = 17) and global OGG1(−/−) (n = 22) mice in the same background (mixture of FBV-C57BL/6). ( E -F ) Global OGG1 (−/−) and wild-type controls were exposed to 500 ppm for 30 min and returned to room air. At 24 h post exposure, OGG1( −/− ) mice had lower mtDNA injury ( E ) and BAL protein levels ( F ) as compared to their wild type controls. Values are means ± SEM; Each symbol represents data from a different mouse. 1-way ANOVA with Tukey post-test.

Article Snippet: The membrane was incubated with OGG1 (Cat No. 15125-1-AP, Proteintech), GAPDH (Cat No. 60004-1-Ig, Proteintech), and β-Tubulin (Cat No. 10094-1-AP, Proteintech), β-Actin (Cat No. 66009-1-Ig, Proteintech), 6∗His-Tag (Cat No. 66005-1-Ig, Proteintech), OGG1 (NB100-106, Novus) in 5 % BSA, 1xTBS, 0.1 % Tween 20 at 4 °C with gentle shaking, for overnight at 4 °C followed by incubation with HRP-conjugated secondary antibodies against mouse (Cat No. 1721011, Bio- Rad Hercules, CA) and rabbit (Cat No. 1706515, Bio-Rad, Hercules, CA) at room temperature for 1 h. Then, images were captured on Syngene Imager by using Supersignal West Dura Extended duration substrate (Cat No. 34076, Thermo scientific).

Techniques: Sterility, Saline, Transgenic Assay, Generated

Journal: Redox Biology

Article Title: Oxidative damage to lung mitochondrial DNA is a key contributor to the development of chemical lung injury

doi: 10.1016/j.redox.2025.103624

Figure Lengend Snippet: Exposure to Cl 2 disrupts the mitochondrial function . Human club-like epithelial cells (H441) were grown in air-liquid interface, and treated with mito OGG1 for 3 h and afterwards exposed to Cl 2 gas at 100 ppm for 10 min and placed in an incubator at 95 % air, 5 % CO 2 . (A) Immunoblot showing exogenous and endogenous OGG1 levels detected by anti-His Tag and OGG1 antibodies, respectively. (B) Immunoblot showing localization of mitoOgg1 fusion protein in H441 cells in cytoplasmic and mitochondrial fractions. ( C ) At 3 h post exposure, H441 cells were stained with Mito tracker (Red) and Mito SOX (green) and examined by confocal fluorescence microscopy. Noticeable co-localization of Mito tracker and Mito SOX (yellow) in Cl 2 exposed cells indicating the presence of mitochondrial reactive oxygen species. H441 cells exposed to Cl 2 (100 ppm for 10 min) and placed in an incubator in 95 % air, 5 % CO2. Post 1hr of Cl 2 exposure, the cells were treated with mitoOGG1 (2.5 μg/ml), added in the apical compartment, and measurements of bioenergetics were performed by Sea Horse 3h later. ( D ) Oxygen consumption rates (OCR; pmol/mg/μg protein) with a SeaHorse at 3 h post exposure. Blue = Air; Red = 3 h post Cl 2 ; Green = 3 h post Cl 2 + mitoOGG1. O = oligomycin; F = FCCP; A = Antimycin A+Rotenone ( E, F, G ) Basal, maximal OCR and ATP linked OCR measurements. Each point represents a value from each well from three different experiments. Mean ± SEM. 1-way ANOVA with Tukey post-test.

Article Snippet: The membrane was incubated with OGG1 (Cat No. 15125-1-AP, Proteintech), GAPDH (Cat No. 60004-1-Ig, Proteintech), and β-Tubulin (Cat No. 10094-1-AP, Proteintech), β-Actin (Cat No. 66009-1-Ig, Proteintech), 6∗His-Tag (Cat No. 66005-1-Ig, Proteintech), OGG1 (NB100-106, Novus) in 5 % BSA, 1xTBS, 0.1 % Tween 20 at 4 °C with gentle shaking, for overnight at 4 °C followed by incubation with HRP-conjugated secondary antibodies against mouse (Cat No. 1721011, Bio- Rad Hercules, CA) and rabbit (Cat No. 1706515, Bio-Rad, Hercules, CA) at room temperature for 1 h. Then, images were captured on Syngene Imager by using Supersignal West Dura Extended duration substrate (Cat No. 34076, Thermo scientific).

Techniques: Western Blot, Staining, Fluorescence, Microscopy

Journal: Cancer immunology research

Article Title: Inhibition of MICA and MICB Shedding Elicits NK cell–mediated Immunity against Tumors Resistant to Cytotoxic T cells

doi: 10.1158/2326-6066.CIR-19-0483

Figure Lengend Snippet: (A) Validation of efficiency of B2M gene inactivation. Control or B2M-KO human A375 melanoma cells were treated with IFNγ for 24 hours and surface expression of HLA-A/B/C was quantified by flow cytometry. MFI = Mean Fluorescence Intensity. (B-D) Control or B2M-KO A375 cells were cultured for 24 hours with MICA/B (7C6-hIgG1) or isotype control antibodies at the indicated concentrations. (B) Quantification of shed MICA released by melanoma cells by sandwich ELISA. (C) MICA/B surface protein on control and B2M-KO melanoma cells was quantified by flow cytometry using PE-conjugated MICA/B mAb 6D4 or an isotype control mAb. (D) Histograms representative of the experiment shown in (C). (E) Effect of human NK cells on A375 cells dependent on MHC-I expression and MICA/B mAb treatment. GFP+ A375 cells (control or B2M-KO) were plated at a density of 5×103 cells per well in a 96-well plate and pre-treated with 7C6-hIgG1 or isotype control mAbs (20 μg/mL) for 24 hours prior to addition of purified human NK cells at different effector to target ratios (0:1, 0.5:1 or 1:1). IL-2 (300 U/mL) was added to support NK-cell survival. The number of GFP+ A375 cells was quantified by imaging cytometry using a Nexcelom Celigo instrument at multiple timepoints over a 72-hour period. Data representative of three independent experiments (A-E). Statistical analyses were performed by two-way ANOVA with Bonferroni’s multiple comparison test (E). Error bars represent standard error (SEM) (A-C and E). *p<0.05, ***p<0.001.

Article Snippet: Diluted cDNA was used for qPCR using TaqMan Gene Expression MasterMix (Life Technologies, 4369016), TaqMan probes (MICA - Hs00741286_m1, MICB - Hs00792952_m1, GAPDH - Hs02786624_g1, ULBP2 - Hs00607609_mH, and RAET1L - Hs04194671_s1) and QuantStudio 6 Flex Real-Time PCR System (ThermoFisher).

Techniques: Biomarker Discovery, Control, Expressing, Flow Cytometry, Fluorescence, Cell Culture, Sandwich ELISA, Purification, Imaging, Cytometry, Comparison

Journal: Cancer immunology research

Article Title: Inhibition of MICA and MICB Shedding Elicits NK cell–mediated Immunity against Tumors Resistant to Cytotoxic T cells

doi: 10.1158/2326-6066.CIR-19-0483

Figure Lengend Snippet: (A) NKG2D ligand mRNA expression following treatment with panobinostat. A375 cells were treated for 24 hours with panobinostat (50 nM), and mRNA was extracted for bulk RNA-seq. mRNA expression for NKG2D ligand and MHC class I genes are shown as ratio (log2 fold-change) for the panobinostat and PBS groups. (B) A375 cells were treated for 24 hours with panobinostat (50 nM) or solvent control (PBS), and the expression of the indicated genes was analyzed by RT-qPCR (triplicates per condition). *p<0.05, **p<0.01, and ***p<0.001, statistical analysis was performed using two-tailed unpaired Student’s t-test with Welch’s correction. Error bars represent standard deviation of three technical replicates. (C) MICA/B surface protein levels following treatment with panobinostat plus MICA/B mAb. A375 cells were incubated with the indicated mAbs (20 μg/mL) and increasing concentrations of panobinostat for 24 hours. MICA/B surface expression (left) and A375 cell viability (right) were quantified by flow cytometry. MICA that was shed into the supernatant was quantified by sandwich ELISA (middle). (D) Representative histograms of the data shown in (C). (E) Treatment of short-term human melanoma cell lines with the combination of panobinostat plus MICA/B mAb. The indicated melanoma cell lines were treated in vitro with the indicated mAbs (20 μg/mL) plus increasing concentrations of panobinostat for 24 hours. MICA/B surface expression was quantified by flow cytometry. Cell lines had different basal and induced expression of MICA/B and were ordered from low to high MICA/B expression. Data representative of three independent experiments (B-C and E). SEM are shown (C and E).

Article Snippet: Diluted cDNA was used for qPCR using TaqMan Gene Expression MasterMix (Life Technologies, 4369016), TaqMan probes (MICA - Hs00741286_m1, MICB - Hs00792952_m1, GAPDH - Hs02786624_g1, ULBP2 - Hs00607609_mH, and RAET1L - Hs04194671_s1) and QuantStudio 6 Flex Real-Time PCR System (ThermoFisher).

Techniques: Expressing, RNA Sequencing, Solvent, Control, Quantitative RT-PCR, Two Tailed Test, Standard Deviation, Incubation, Flow Cytometry, Sandwich ELISA, In Vitro

Journal: Cancer immunology research

Article Title: Inhibition of MICA and MICB Shedding Elicits NK cell–mediated Immunity against Tumors Resistant to Cytotoxic T cells

doi: 10.1158/2326-6066.CIR-19-0483

Figure Lengend Snippet: (A) B16F10-MICA cells (control, B2m-KO, or Jak1-KO) were treated for 24 hours with IFNγ (10 ng/mL) or solvent control (PBS), and subsequently surface level of H-2Kb was analyzed by flow cytometry. (B) MICA/B mAb treatment effects on established metastases with inactivating mutations in B2m or Jak1 genes. B16F10-MICA cells (7×105 control, B2m-KO or Jak1-KO tumor cells) were injected i.v. into B cell–deficient (Ighm–/–) mice. On day 7, a subset of mice was euthanized for quantification of metastases, while the remaining mice were treated with 7C6-mIgG2a or control mAbs (200 μg i.p. on days 7, 8, and 12). On day 14, lung surface metastases were counted under a stereomicroscope. Each dot represents one mouse and error bars indicate standard deviation (SD). (C) Impact of MICA/B mAb treatment on survival of mice with B2m or Jak1 deficient melanoma metastases. WT mice (Ighm+/+) were inoculated i.v. with 2 × 105 control, B2m-KO, or Jak1-KO B16F10-MICA cells. Mice received 7C6-mIgG2a or isotype control mAbs on days 1 and 2, and mouse survival was recorded. The number of mice per group is as follows: Control (n=10), B2m-KO (n=20), and Jak1-KO (n=20). Data representative of three independent experiments (A) or pooled from three (B) or two (C) independent experiments. Statistical analyses were performed by two-tailed unpaired Student’s t-tests (B), and Log-rank (Mantel-Cox) test (C). *p<0.05, **p<0.01, ***p<0.001.

Article Snippet: Diluted cDNA was used for qPCR using TaqMan Gene Expression MasterMix (Life Technologies, 4369016), TaqMan probes (MICA - Hs00741286_m1, MICB - Hs00792952_m1, GAPDH - Hs02786624_g1, ULBP2 - Hs00607609_mH, and RAET1L - Hs04194671_s1) and QuantStudio 6 Flex Real-Time PCR System (ThermoFisher).

Techniques: Control, Solvent, Flow Cytometry, Injection, Standard Deviation, Two Tailed Test

Journal: Cancer immunology research

Article Title: Inhibition of MICA and MICB Shedding Elicits NK cell–mediated Immunity against Tumors Resistant to Cytotoxic T cells

doi: 10.1158/2326-6066.CIR-19-0483

Figure Lengend Snippet: (A) Expression of MHC-I by LLC1-MICA cells. Control or B2m-KO LLC1-MICA cells were either stimulated with IFNγ (10 ng/mL) or solvent control (PBS) for 24 hours. Surface H-2Kb protein was quantified by flow cytometry. (B) MICA/B mAb treatment of lung metastases formed by LLC1 lung cancer cells. WT C57BL6/J mice were inoculated i.v. with 1×106 (1M) or 1.5×106 (1.5M) LLC1-MICA tumor cells (control or B2m-KO). On day 2 following tumor cell inoculation, mice were treated with indicated mAb (200 μg i.p.); additional treatments were given on day 3 and then once per week. Lung metastases were counted on day 14. (C) MICA/B mAb treatment of LLC1-MICA metastases in mice reconstituted with allogeneic or syngeneic NK cells. Rag2–/–Il2rg–/– double-knockout mice were injected with NK cells (2×105 cells) from CB6F1/J mice or C56BL/6 mice, which were allogeneic or syngeneic to LLC1 cells, respectively. A third group of Rag2–/–Il2rg–/– mice did not receive NK cells. LLC1-MICA tumor cells (7×105) were injected i.v. 24 hours following NK-cell transfer. On days 2, 3, and then once per week following tumor cell inoculation, mice were treated with the indicated antibodies (200 μg). Metastases were counted on day 14. Data representative of three independent experiments (A) or pooled from three (B) or two (C) independent experiments. Statistical analyses were performed by two-tailed unpaired Student’s t-test (B - C). *p<0.05, **p<0.01, ***p<0.001. Each dot represents one mouse, and SD is shown (B-C).

Article Snippet: Diluted cDNA was used for qPCR using TaqMan Gene Expression MasterMix (Life Technologies, 4369016), TaqMan probes (MICA - Hs00741286_m1, MICB - Hs00792952_m1, GAPDH - Hs02786624_g1, ULBP2 - Hs00607609_mH, and RAET1L - Hs04194671_s1) and QuantStudio 6 Flex Real-Time PCR System (ThermoFisher).

Techniques: Expressing, Control, Solvent, Flow Cytometry, Double Knockout, Injection, Two Tailed Test

Journal: Cancer immunology research

Article Title: Inhibition of MICA and MICB Shedding Elicits NK cell–mediated Immunity against Tumors Resistant to Cytotoxic T cells

doi: 10.1158/2326-6066.CIR-19-0483

Figure Lengend Snippet: (A) Wild-type (WT) C57BL/6 mice were inoculated i.v. with 7×105 B16F10-MICA cells (control, B2m-KO, or Jak1-KO). Mice were treated with 7C6-mIgG2a or isotype control mAbs (200 μg) one day later, as well as on days 2 and 7. CD8+ T-cell depletion was performed by injection of anti-CD8β, whereas NK-cell depletion was performed by injection of 100μg of anti-asialo GM1 (anti-asGM1) or anti-NK1.1 on days −1, 0, and 7 after tumor cell inoculation. Control mice received an isotype control antibody. Lung surface metastases were quantified on day 14 following tumor inoculation. (B) Analysis of NK-cell infiltration into lung tissue. Tumor injection and mAb treatment were done as described in (A), with tumor cells that expressed ZsGreen to enable their identification by flow cytometry. On day 12 following tumor cell inoculation, mice were injected i.v. with APC-conjugated anti-CD45.2 to distinguish blood and tissue-infiltrating NK cells, as reported previously (23). Lung-infiltrating NK cells were identified as CD3ε–TCRβ–NK1.1+CD49b+EOMES+ viable cells with low staining for CD45.2-APC (injected i.v.) but high staining for CD45.2-PE-CY7 (added to cell suspension). The ratio of NK cells to ZsGreen+ B16F10-MICA cells is shown. (C) Numbers of ZsGreen+ B16F10-MICA cells and (D) lung-infiltrating NK cells for the indicated genotypes and treatment groups for the experiment described in (B). EOMES labeling was used to differentiate NK cells from ILC1 cells. Data pooled from two independent experiments (A-D). Statistical analyses were performed using two-way ANOVA with Bonferroni’s posthoc test (A) or two-tailed unpaired Student’s t-test (B-D), *p<0.05, **p,0.01, ***p<0.001. SD (A-B) and SEM (C-D) are shown.

Article Snippet: Diluted cDNA was used for qPCR using TaqMan Gene Expression MasterMix (Life Technologies, 4369016), TaqMan probes (MICA - Hs00741286_m1, MICB - Hs00792952_m1, GAPDH - Hs02786624_g1, ULBP2 - Hs00607609_mH, and RAET1L - Hs04194671_s1) and QuantStudio 6 Flex Real-Time PCR System (ThermoFisher).

Techniques: Control, Injection, Flow Cytometry, Staining, Suspension, Labeling, Two Tailed Test

Journal: Cancer immunology research

Article Title: Inhibition of MICA and MICB Shedding Elicits NK cell–mediated Immunity against Tumors Resistant to Cytotoxic T cells

doi: 10.1158/2326-6066.CIR-19-0483

Figure Lengend Snippet: (A) In vivo effects of panobinostat plus MICA/B mAb treatment on MICA/B surface protein in metastases formed by human melanoma cells. NSG mice were inoculated i.v. with 1×106 ZsGreen+ A375 cells. Two weeks later, mice were treated on two subsequent days with the indicated mAbs (200 μg) +/− panobinostat (10 mg/kg). 24 hours following the last treatment, MICA/B surface expression was analyzed on tumor cells in lung metastases (large, viable, ZsGreen+CD45– cells). (B-C) NSG mice were reconstituted with purified human NK cells (2×106 i.v.) expanded in vitro for 1–3 weeks. In vivo survival of NK cells was supported by simultaneous administration of IL2 (7.5× 104 units) via intraperitoneal injection. On day 1, mice were inoculated i.v. with control or B2M-KO A375 cells (5×105). On days 2 and 3, mice received another dose of IL-2, the indicated mAbs (200 μg) +/− panobinostat (10 mg/kg); on day 3 an additional dose of NK cells was also administered. On day 14, the number of lung surface metastases was counted. (B) Illustration of experimental design and (C) quantification of lung surface metastases. Data pooled from two independent experiments (A and C). Statistical analyses were performed by two-tailed unpaired Student’s t-test (A) and two-way ANOVA, Bonferroni’s post-hoc test (C), *p<0.05, **p<0.01, ***p<0.001. Error bars represent SD (A and C).

Article Snippet: Diluted cDNA was used for qPCR using TaqMan Gene Expression MasterMix (Life Technologies, 4369016), TaqMan probes (MICA - Hs00741286_m1, MICB - Hs00792952_m1, GAPDH - Hs02786624_g1, ULBP2 - Hs00607609_mH, and RAET1L - Hs04194671_s1) and QuantStudio 6 Flex Real-Time PCR System (ThermoFisher).

Techniques: In Vivo, Expressing, Purification, In Vitro, Injection, Control, Two Tailed Test

Journal: bioRxiv

Article Title: Computational modeling and preclinical validation support targeting somatic instability for Huntington’s disease treatment

doi: 10.64898/2026.01.06.697909

Figure Lengend Snippet: (A-B) Still frames from an animation of untreated (A) and treated (B) HD patients are shown from example simulations of 3000 MSNs that depict somatic CAG repeat expansion in individual MSNs at 20-year intervals from 1-100 years. (A) Each point represents an untreated MSN and simulated CAG length (x-axis). The red line represents the 150 CAG threshold for transcriptionally healthy MSNs (<150 CAG; green) and dysregulated unhealthy MSNs (>150 CAG; red) progressing toward cell death (>300 CAGs). (B) Simulated MSNs are shown with SI-targeted therapy administration at 32 years old and 50% of MSNs targeted with 50% MSH3 knockdown. Targeted MSNs are circled in pink. (C) The percentage of untreated healthy MSNs (<150 CAGs) are tracked and plotted across a simulated lifetime (0-100 years). Shaded background panels represent the indicated HD-ISS stage, age (shown in red) when a HD-ISS stage is reached, and dotted lines represent a HD milestone event (measurable volumetric change and CAP-100 equivalent age). (D) A treated purple line is compared to the untreated grey line from (C) and tracks the percentage of healthy treated MSNs after administration at 32 years old. (E-G) The model tracks therapeutic benefit by determining the age and healthy MSN number coinciding with a HD landmark event (onset of striatum volume change, motor symptoms, or HD-ISS stage). (H) The model enables prediction of cUHDRS change from baseline between simulated treated (purple dotted line) and untreated Enroll-HD natural history cohorts (grey dotted line).

Article Snippet: Total RNA was extracted from NHP brain samples using the Quick-DNA/RNA Miniprep Plus kit (Zymo Research D7003) and quantified with the Qubit BR Assay kit (Life Technologies Q10210). cDNA was generated from 500 ng total RNA with Maxima H minus reverse transcriptase (Thermo Scientific EP0752) and random hexamers (Life Technologies SO142). qPCR was performed with Luna Primer Probe 2X Master Mix (NEB M3004) including primer probe sets for rhesus MSH3 (Applied Biosystems Rh00989001_m1) and TBP (Applied Biosystems Rh00427620_m1).

Techniques: Knockdown

Journal: bioRxiv

Article Title: Computational modeling and preclinical validation support targeting somatic instability for Huntington’s disease treatment

doi: 10.64898/2026.01.06.697909

Figure Lengend Snippet: (A-C) Heatmaps depicting the simulated therapeutic benefit for a SI-lowering therapy in a range of treated patients with germline CAG repeat lengths from 37-60 CAGs (y-axis) and ages at treatment from 5-60 years of age (x-axis). The degrees of predicted therapeutic benefit as measured by years of delay to CAP-100 equivalent are depicted in the heatmaps (shown in the heatmap key; less than 5 years of benefit shown in grey, CAP-100 equivalent never reached shown in purple, and benefit between >5 and <50 years shown by the white to pink gradient). (A) Heatmap depicting the simulated range of therapeutic benefit achieved by targeting 25% of MSNs with 25% MSH3 knockdown per MSN. (B) Heatmaps depicting the range of therapeutic benefit with 50% MSH3 knockdown per MSN and a range of MSN targeting percentages (50%, 75%, and 100%). (C) Heatmaps depicting the range of therapeutic benefit with targeting 50% of MSNs and a range of MSH3 knockdown percentages (50%, 75%, and 100%) per MSN.

Article Snippet: Total RNA was extracted from NHP brain samples using the Quick-DNA/RNA Miniprep Plus kit (Zymo Research D7003) and quantified with the Qubit BR Assay kit (Life Technologies Q10210). cDNA was generated from 500 ng total RNA with Maxima H minus reverse transcriptase (Thermo Scientific EP0752) and random hexamers (Life Technologies SO142). qPCR was performed with Luna Primer Probe 2X Master Mix (NEB M3004) including primer probe sets for rhesus MSH3 (Applied Biosystems Rh00989001_m1) and TBP (Applied Biosystems Rh00427620_m1).

Techniques: Knockdown

Journal: bioRxiv

Article Title: Computational modeling and preclinical validation support targeting somatic instability for Huntington’s disease treatment

doi: 10.64898/2026.01.06.697909

Figure Lengend Snippet: (A) Study design for the nonhuman primate dose range finding study in rhesus macaques. (B) AAV vector biodistribution (vector genomes per diploid genome) in the globus pallidus (GP), caudate nucleus, and putamen of vehicle and treated rhesus macaques by droplet digital PCR (ddPCR) after bilateral intraparenchymal (IPa) GP administration of AAV-DB-3.miMSH3-06. (C) miMSH3 expression levels normalized to copies per µg of RNA by stem-loop RT-qPCR. (D) MSH3 mRNA levels normalized to TBP and relative to vehicle control by RT-qPCR. (E) MSH3 protein levels relative to vehicle control by Jess capillary-electrophoresis immunoassay. Data are shown as group mean (N=2) with data points from each animal. Panel A was created using BioRender.com.

Article Snippet: Total RNA was extracted from NHP brain samples using the Quick-DNA/RNA Miniprep Plus kit (Zymo Research D7003) and quantified with the Qubit BR Assay kit (Life Technologies Q10210). cDNA was generated from 500 ng total RNA with Maxima H minus reverse transcriptase (Thermo Scientific EP0752) and random hexamers (Life Technologies SO142). qPCR was performed with Luna Primer Probe 2X Master Mix (NEB M3004) including primer probe sets for rhesus MSH3 (Applied Biosystems Rh00989001_m1) and TBP (Applied Biosystems Rh00427620_m1).

Techniques: Plasmid Preparation, Digital PCR, Expressing, Quantitative RT-PCR, Control, Electrophoresis

Journal: bioRxiv

Article Title: Computational modeling and preclinical validation support targeting somatic instability for Huntington’s disease treatment

doi: 10.64898/2026.01.06.697909

Figure Lengend Snippet: (A-B) Representative RNAscope FISH confocal microscopy images of MSH3 mRNA puncta (green) in PPP1R1B+ MSNs (magenta) in the caudate (A) and putamen (B) of NHPs. Hoechst stain (blue) was used to counterstain nuclei. (C) Cytoplasmic MSH3 mRNA puncta count in PPP1R1B+ MSNs of the striatum (caudate and putamen) were determined with an automated cell segmentation and puncta counting algorithm. The percentages of cytoplasmic MSH3 mRNA knockdown (KD) levels in all MSNs profiled in the caudate and putamen relative to vehicle control are shown above each treatment group. Total MSN counts are displayed below each group. Data are shown as group mean (N=2) with data points from each animal.

Article Snippet: Total RNA was extracted from NHP brain samples using the Quick-DNA/RNA Miniprep Plus kit (Zymo Research D7003) and quantified with the Qubit BR Assay kit (Life Technologies Q10210). cDNA was generated from 500 ng total RNA with Maxima H minus reverse transcriptase (Thermo Scientific EP0752) and random hexamers (Life Technologies SO142). qPCR was performed with Luna Primer Probe 2X Master Mix (NEB M3004) including primer probe sets for rhesus MSH3 (Applied Biosystems Rh00989001_m1) and TBP (Applied Biosystems Rh00427620_m1).

Techniques: RNAscope, Confocal Microscopy, Staining, Knockdown, Control

Journal: bioRxiv

Article Title: Computational modeling and preclinical validation support targeting somatic instability for Huntington’s disease treatment

doi: 10.64898/2026.01.06.697909

Figure Lengend Snippet: (A-D) Heatmaps depicting the simulated range of therapeutic benefit for patients treated with an SI-lowering AAV gene therapy over a lifetime of 100 years. Each heatmap represents a range of patients with germline CAG lengths from 40-50 CAG repeats (y-axis) and age at treatment (x-axis). The degrees of predicted therapeutic benefit as measured by years of delay to CAP-100 equivalent are depicted in the heatmaps (shown in the heatmap key; less than 5 years of benefit shown in grey, CAP-100 equivalent was never reached shown in purple, and benefit between >5 and <50 years shown by the gradient from white to pink). (A) The range of therapeutic benefit achieved after simulating 50% of MSNs transduced and 50% MSH3 KD per transduced MSN. This threshold is denoted by black bordered heatmap cells in all four heatmaps. (B-D) Heatmaps depict the predicted range of therapeutic benefit for HD patients treated with the NHP DRF dose equivalents of AAV-DB-3.miMSH3 at the low dose (1.5×10 11 vg) (B) , mid dose (8.2×10 11 vg) (C) , and high dose (3.2×10 12 vg) (D) based on the RNAscope FISH MSH3 mRNA KD levels detected in the NHP caudate and putamen . CAG repeat length incidence is shown by the red to yellow color gradient column . (E) To characterize predicted therapeutic performance using cUHDRS across the observed percentages of MSH3 knockdown, we simulated these therapeutic performance levels in a hypothetical patient with 42 CAGs treated at the age of 46.

Article Snippet: Total RNA was extracted from NHP brain samples using the Quick-DNA/RNA Miniprep Plus kit (Zymo Research D7003) and quantified with the Qubit BR Assay kit (Life Technologies Q10210). cDNA was generated from 500 ng total RNA with Maxima H minus reverse transcriptase (Thermo Scientific EP0752) and random hexamers (Life Technologies SO142). qPCR was performed with Luna Primer Probe 2X Master Mix (NEB M3004) including primer probe sets for rhesus MSH3 (Applied Biosystems Rh00989001_m1) and TBP (Applied Biosystems Rh00427620_m1).

Techniques: RNAscope, Knockdown

Journal: bioRxiv

Article Title: Computational modeling and preclinical validation support targeting somatic instability for Huntington’s disease treatment

doi: 10.64898/2026.01.06.697909

Figure Lengend Snippet: (A) Study design for the pharmacology study in heterozygous HdhQ111 KI mice. (B) Msh3 mRNA levels in the striatum of untreated and treated HdhQ111 mice by RT-qPCR. Msh3 mRNA levels were normalized to ActB mRNA and shown as the group mean relative to vehicle control (N=7, 8 week untreated; N=7, 24 week untreated; N=9, vehicle; N=10, 5×10 9 vg; N=10, 1.5×10 10 vg; N=10, 5×10 10 vg; N=8, 1.5×10 11 vg; one-way ANOVA with Dunnett’s post hoc analysis, ** p < 0.01, *** p < 0.001, **** p < 0.0001). (C) Fragment analysis traces shown in blue depict the intensity of CAG repeat length alleles in untreated, vehicle and AAV-DB-3.miMSH3-06 treated mice by PCR and capillary electrophoresis. (D) Somatic instability index in untreated and treated mice (N=10/group; one-way ANOVA with Dunnett’s post hoc analysis, **** p < 0.0001). Data are shown as the mean and error bars represent SD. The percent reduction in SI relative to vehicle control is shown above each treatment group. Panel A was created using BioRender.com.

Article Snippet: Total RNA was extracted from NHP brain samples using the Quick-DNA/RNA Miniprep Plus kit (Zymo Research D7003) and quantified with the Qubit BR Assay kit (Life Technologies Q10210). cDNA was generated from 500 ng total RNA with Maxima H minus reverse transcriptase (Thermo Scientific EP0752) and random hexamers (Life Technologies SO142). qPCR was performed with Luna Primer Probe 2X Master Mix (NEB M3004) including primer probe sets for rhesus MSH3 (Applied Biosystems Rh00989001_m1) and TBP (Applied Biosystems Rh00427620_m1).

Techniques: Quantitative RT-PCR, Control, Electrophoresis

Journal: Nucleic Acids Research

Article Title: MutSβ and histone deacetylase complexes promote expansions of trinucleotide repeats in human cells

doi: 10.1093/nar/gks810

Figure Lengend Snippet: ( A ) MSH2 and MSH3, but not MSH6, are enriched at the (CTG) 22 repeat tract in human SVG-A cells. Cells were transfected with a shuttle vector containing either a (CTG) 22 repeat tract (red bars) or a corresponding randomized (C,T,G) 22 sequence as a control (white bars). ChIP reactions were performed subsequently with antibodies specific for MSH2, MSH3 or MSH6, while background signals were assessed using a FLAG tag antibody. Real-time PCR signals for occupancy of MSH2, MSH3 and MSH6 are presented as fold enrichment over background signals. Error bars, ±SEM; * P < 0.05 compared to randomized (C,T,G) 22 control; n = 5. ( B ) Schematic showing position of real-time PCR primers for ChIP experiments, relative to the (CTG) 22 tract or randomized (C,T,G) 22 sequence. The ChIP amplicon is 89 bp, with 69 bp of flanking sequence between the amplicon and the repeat tract or control sequence.

Article Snippet: IP reactions were set up with 5 μg MSH2 antibody (ab16833, Abcam), 5 μg MSH3 antibody (ab74607) and 2.5 μg MSH6 antibody (sc-10798, Santa Cruz), while a FLAG antibody (A8592, Sigma) was used as a negative control to assess levels of background.

Techniques: Transfection, Plasmid Preparation, Sequencing, Control, FLAG-tag, Real-time Polymerase Chain Reaction, Amplification

Journal: Nucleic Acids Research

Article Title: MutSβ and histone deacetylase complexes promote expansions of trinucleotide repeats in human cells

doi: 10.1093/nar/gks810

Figure Lengend Snippet: Expansions are suppressed following siRNA knockdown of MSH2 and MSH3, but not by knockdown of MSH6, CtIP or Mre11. ( A ) Expansion frequencies subsequent to treatment with MSH2 siRNA, MSH3 siRNA, MSH6 siRNA, CtIP individual siRNAs (denoted #1 and #2) and Mre11 siRNA. All frequencies are normalized to scrambled siRNA, denoted as ‘Scr’ (white bars). Error bars denote ±1 SEM; * P < 0.05 compared to scrambled siRNA control; n = 4 for MSH2 and MSH6 siRNA, n = 3 for MSH3, CtIP and Mre11 siRNA. ( B ) Quantification of protein levels following knockdown, normalized to actin and to the scrambled siRNA control. Error bars denote ±1 SEM; n = 3.

Article Snippet: IP reactions were set up with 5 μg MSH2 antibody (ab16833, Abcam), 5 μg MSH3 antibody (ab74607) and 2.5 μg MSH6 antibody (sc-10798, Santa Cruz), while a FLAG antibody (A8592, Sigma) was used as a negative control to assess levels of background.

Techniques: Knockdown, Control

Journal: Nucleic Acids Research

Article Title: MutSβ and histone deacetylase complexes promote expansions of trinucleotide repeats in human cells

doi: 10.1093/nar/gks810

Figure Lengend Snippet: RNAi knockdown of HDAC3 does not affect occupancy of MSH2, MSH3 or MSH6 at (CTG) 22 repeat tracts, or protein expression of MSH2 or MSH3, in SVG-A cells. ( A ) SVG-A cells were transiently transfected with a (CTG) 22 -repeat containing shuttle vector, in the presence of scrambled siRNA (white bars) or HDAC3 siRNA (navy bars). ChIP reactions were performed subsequently with antibodies specific for MSH2, MSH3 or MSH6, while background signals were assessed using a FLAG tag antibody. Signals for occupancy of MSH2, MSH3 and MSH6 are presented as fold enrichment over background signals. Error bars, ±SEM; * P < 0.05 compared to control; n = 5. ( B ) Representative immunoblots for MSH2 and MSH3 protein expression in SVG-A cells following transfection with scrambled siRNA or HDAC3 siRNA. For assessment of MSH2 and MSH3 protein expression, α-tubulin was used as a loading control. For assessment of HDAC3 protein expression, actin was used as a loading control.

Article Snippet: IP reactions were set up with 5 μg MSH2 antibody (ab16833, Abcam), 5 μg MSH3 antibody (ab74607) and 2.5 μg MSH6 antibody (sc-10798, Santa Cruz), while a FLAG antibody (A8592, Sigma) was used as a negative control to assess levels of background.

Techniques: Knockdown, Expressing, Transfection, Plasmid Preparation, FLAG-tag, Control, Western Blot

Journal: bioRxiv

Article Title: Somatic CAG expansion in Huntington’s disease is dependent on the MLH3 endonuclease domain, which can be excluded via MLH3 splice redirection to suppress expansion

doi: 10.1101/2020.10.26.356238

Figure Lengend Snippet: ( A ) Schematic representation of MLH3 protein and its functional domains, as well as mutation of the endonucleolytic motif in Mlh3 DN mice (adapted from Toledo et al . 2019 ). ( B ) Quantification of HTT CAG expansions in striatum, liver, and kidney at 6 months of age in Htt Q111 knock-in mice that are either wild type ( Mlh3 WT/WT , CAG 114-121), heterozygous ( Mlh3 WT/DN , CAG 116-121), or homozygous ( Mlh3 DN/DN , CAG 116-121) for the D1185N mutation shows that an intact MLH3 endonuclease domain is required for somatic CAG expansion. Striatum and liver, n = 3; kidney, n = 2-3; error bars represent standard deviation of the mean; two-way ANOVA (with genotype and tissue as variables) with Tukey multiple comparisons post hoc correction. Genotype effect: ns , not significant; ***, p ≤ 0.001; ****, p ≤ 0.0001. ( C ) Representative GeneMapper traces across different tissues at 6 months of age in Htt Q111 knock-in mice (CAG 121) with different Mlh3 genotypes.

Article Snippet: The assay used for the Mlh3 splice variant 1 detects the splice junction between exon 6 and exon 7 (Mm00520819_m1, FAM-labeled probe).

Techniques: Functional Assay, Mutagenesis, Knock-In, Standard Deviation

Journal: bioRxiv

Article Title: Somatic CAG expansion in Huntington’s disease is dependent on the MLH3 endonuclease domain, which can be excluded via MLH3 splice redirection to suppress expansion

doi: 10.1101/2020.10.26.356238

Figure Lengend Snippet: ( A ) Schematic representation of mouse Mlh3 splice redirection with splice switching oligonucleotides (SSOs). SSOs were designed to bind the intron–exon junctions in mouse Mlh3 pre-mRNA at either the splice acceptor (mMlh3ac5) or donor (mMlh3dr7) regions of the endonuclease-coding exon 6, which is analogous to the human MLH3 exon 7, inducing exon skipping and preferential production of Mlh3 splice variant 2, which lacks the endonuclease domain. ( B ) Summary of Mlh3 SSOs systemic treatment in HD mice. Htt Q111 mice (two males and two females per group) were treated with either 25 mg/kg Mlh3 SSOs (mMLH3ac5 and mMLH3dr7, 1:1 ratio; CAG 106-109), or PBS (CAG 104-108), by tail vein injection. Treatment was initiated at 4 weeks of age and injections were performed three times per week (every 48-72hr). Weight measurements were taken at the beginning of each week to determine weekly weight-adjusted doses. Treatment was stopped at 12 weeks of age, with tissue collection performed 24 hours after the final injection. ( C ) RT-PCR analyses of Mlh3 mRNA splice variants in 12-week-old Htt Q111 mice, following treatment with Mlh3 SSOs, reveals successful splice redirection from splice variant 1 (Var1) to variant 2 (Var2) in the kidney (K), and to a lesser extent in the liver (L). ( D ) Quantification of CAG expansion indices reveals statistically significant reductions in somatic HTT CAG expansion in kidney and, to a lesser extent, in liver of mice that received Mlh3 SSO treatment when compared to PBS. n = 4; error bars represent standard deviation of the mean; 2-tailed unpaired t test: ns , not significant; *, p ≤ 0.05; ****, p ≤ 0.0001. ( E ) Representative GeneMapper traces of somatic HTT CAG repeat size distributions across different tissues at 12 weeks of age in Htt Q111 knock-in mice (CAG 108), following PBS or Mlh3 SSO treatment.

Article Snippet: The assay used for the Mlh3 splice variant 1 detects the splice junction between exon 6 and exon 7 (Mm00520819_m1, FAM-labeled probe).

Techniques: Variant Assay, Injection, Reverse Transcription Polymerase Chain Reaction, Standard Deviation, Knock-In

Journal: bioRxiv

Article Title: Somatic CAG expansion in Huntington’s disease is dependent on the MLH3 endonuclease domain, which can be excluded via MLH3 splice redirection to suppress expansion

doi: 10.1101/2020.10.26.356238

Figure Lengend Snippet: ( A ) Schematic representation of human MLH3 splice redirection with splice switching oligonucleotides (SSOs) (adapted from Halabi et al . 2018 ). SSOs were designed to bind the intron–exon junctions in human MLH3 pre-mRNA at either the splice acceptor (MLH3ac6) or donor (MLH3dnr8) regions of the endonuclease-coding exon 7, inducing exon skipping and preferential production of MLH3 splice variant 2, which lacks the endonuclease domain. ( B ) RT-PCR analyses of MLH3 mRNA splice variants in HD patient-derived primary fibroblasts (GM09197, CAG ~180/18) following treatment with 500 nM of either scrambled control vivo-morpholino oligonucleotide (Scr) or MLH3 SSOs (SSO) for 48 hours, alongside untreated controls (Unt). Splice redirection from predominantly splice variant 1 (Var1) to predominantly splice variant 2 (Var2), which lacks exon 7 and the endonuclease domain, was efficiently achieved with MLH3 SSOs treatment. No splice redirection was observed in scrambled oligotreated cells, which were identical to untreated cells. ( C ) In order to induce CAG expansion in HD patient fibroblasts (GM09197, CAG ~180/18), cells were initially treated with lentiviral particles for stable ectopic expression of MSH3. Cells were then cultured to confluency, to inhibit replication, and treated twice weekly with 500 nM of MLH3 SSOs (MLH3acr6 and MLH3dnr8, 1:1 ratio, 250 nM each), or left untreated, for a total of six weeks when cells were harvested for HTT CAG instability analysis. ( D ) Quantification of average HTT CAG gain during the 6 week-long treatment (relative to average CAG prior to treatment, “ t = 0”, n = 4) reveals a potent inhibition of CAG expansion by MLH3 SSOs (average gain of 0.6 CAGs, n = 10), when compared to untreated cells (average gain of 3.0 CAGs, n = 10). Error bars represent standard deviation of the mean; one-way ANOVA with Tukey multiple comparisons post hoc correction; ****, p ≤ 0.0001. ( E ) Representative GeneMapper traces of HTT CAG repeat size distributions from GM09197 HD patient fibroblasts before ( t = 0) and after ( t = 6 weeks) treatment with MLH3 SSOs (CAG gain = 0.7), or no treatment (CAG gain = 3.6).

Article Snippet: The assay used for the Mlh3 splice variant 1 detects the splice junction between exon 6 and exon 7 (Mm00520819_m1, FAM-labeled probe).

Techniques: Variant Assay, Reverse Transcription Polymerase Chain Reaction, Derivative Assay, Control, Expressing, Cell Culture, Inhibition, Standard Deviation

Journal: Nature Communications

Article Title: MTH1 protects platelet mitochondria from oxidative damage and regulates platelet function and thrombosis

doi: 10.1038/s41467-023-40600-7

Figure Lengend Snippet: Human or wild-type mouse platelets were isolated to measure MTH1 expression by western blot with three different antibodies using human multiple myeloma (MM) cells or mouse acute myeloid leukemia (AML) cells as positive control (representative of three independent experiments) ( a ). b MTH1 expression in MTH1-deficient platelets was measured by western blot using MTH1 antibody (Santa Cruz). c Platelet cytosol and mitochondria were isolated from wild-type mice to measure the expression of MTH1 (MTH1 antibody from Santa Cruz), VDAC (VDAC antibody from Abcam) or GAPDH (representative of three independent experiments). d Platelets were isolated from wild-type mouse and then fixed followed by labeling with MTH1 antibody and then with the secondary antibody to evaluate the localization of MTH1 using immune electron microscopy ( n = 3 independent isolated platelets). M indicates mitochondria and arrows indicates the positive expression of MTH1. Left panel: ×12,000 and right panel: ×30,000. e Tail bleeding time (mean, n = 10 independent animals, two-tailed Mann–Whitney test) and f arterial thrombus formation (mean, n = 10 independent animals, two-tailed Mann-Whitney test). Representative image of thrombus formation at 5, 15 and 20 min was shown. The dotted lines indicate arterial vessel walls. Scale bar = 200 μm. g Venous thrombosis evaluation. Mice underwent ligation of inferior vena cava (IVC) to induce venous thrombosis and IVC samples were collected after 24 h of ligation to measure thrombus length and weight (mean, n = 10 independent animals, two-tailed Mann–Whitney test). The representative image of the venous thrombi was shown in the right panel. Scale bar = 1 mm.

Article Snippet: Human or wild-type mouse platelets were isolated to measure MTH1 expression by western blot with three different antibodies using human multiple myeloma (MM) cells or mouse acute myeloid leukemia (AML) cells as positive control (representative of three independent experiments) ( a ). b MTH1 expression in MTH1-deficient platelets was measured by western blot using MTH1 antibody (Santa Cruz). c Platelet cytosol and mitochondria were isolated from wild-type mice to measure the expression of MTH1 (MTH1 antibody from Santa Cruz), VDAC (VDAC antibody from Abcam) or GAPDH (representative of three independent experiments). d Platelets were isolated from wild-type mouse and then fixed followed by labeling with MTH1 antibody and then with the secondary antibody to evaluate the localization of MTH1 using immune electron microscopy ( n = 3 independent isolated platelets).

Techniques: Isolation, Expressing, Western Blot, Positive Control, Labeling, Electron Microscopy, Two Tailed Test, MANN-WHITNEY, Ligation

Journal: Nature Communications

Article Title: MTH1 protects platelet mitochondria from oxidative damage and regulates platelet function and thrombosis

doi: 10.1038/s41467-023-40600-7

Figure Lengend Snippet: Washed platelets (200 × 10 9 /l) from MTH1 fl/fl or MTH1 -/- mice were stimulated with CRP (0.1 μg/ml) (mean ± SE, n = 3 independent isolated platelets) ( a ), thrombin (0.01 U/ml) (mean ± SE, n = 4 independent isolated platelets) ( b ), U46619 (0.3 μM) (mean ± SE, n = 3 independent isolated platelets) ( c ) followed by analysis of platelet aggregation and ATP release (reflecting dense granule secretion) in a Lumi-Aggregometer Model 700 (two-tailed unpaired Student’s t test). Washed platelets were stimulated with CRP or thrombin to measure integrin αIIbβ3 activation (presented by JON/A binding) (mean ± SE, n = 4 independent isolated platelets, two-way ANOVA with Sidak multiple comparisons test) ( d ) and phosphatidylserine exposure (presented by Annexin-V binding) (mean ± SE, n = 3 independent isolated platelets, two-way ANOVA with Sidak multiple comparisons test) ( e ) by flow cytometry or calcium mobilization using Fluo-4 AM by a microplate reader (mean ± SE, n = 3 independent isolated platelets, two-tailed unpaired Student’s t test) ( f ). g Platelet aggregation in response to thrombin (0.01 U/ml) after addition of apyrase (0.25 U/ml) or ADP (1 μM) (mean ± SE, n = 3 independent isolated platelets, one-way ANOVA with Tukey multiple comparisons test). h Mitochondrial ATP production was measured using the Biotracker dye in thrombin-stimulated platelets (mean ± SE, n = 4 independent isolated platelets, two-way ANOVA with Sidak multiple comparisons test).

Article Snippet: Human or wild-type mouse platelets were isolated to measure MTH1 expression by western blot with three different antibodies using human multiple myeloma (MM) cells or mouse acute myeloid leukemia (AML) cells as positive control (representative of three independent experiments) ( a ). b MTH1 expression in MTH1-deficient platelets was measured by western blot using MTH1 antibody (Santa Cruz). c Platelet cytosol and mitochondria were isolated from wild-type mice to measure the expression of MTH1 (MTH1 antibody from Santa Cruz), VDAC (VDAC antibody from Abcam) or GAPDH (representative of three independent experiments). d Platelets were isolated from wild-type mouse and then fixed followed by labeling with MTH1 antibody and then with the secondary antibody to evaluate the localization of MTH1 using immune electron microscopy ( n = 3 independent isolated platelets).

Techniques: Isolation, Two Tailed Test, Activation Assay, Binding Assay, Flow Cytometry

Journal: Nature Communications

Article Title: MTH1 protects platelet mitochondria from oxidative damage and regulates platelet function and thrombosis

doi: 10.1038/s41467-023-40600-7

Figure Lengend Snippet: a Accumulation of 8-oxo-dG in mitochondria of platelets from MTH1 fl/fl or MTH1 −/− mice after stimulation by thrombin (1 U/ml) or CRP (5 μg/ml) (mean ± SE, n = 3 independent isolated platelets, two-way ANOVA with Sidak multiple comparisons test). b Washed platelets were loaded with MitoSOX Red (5 μM) for 10 min and then stimulated with thrombin (0.25 U/ml) for 3 min to measure mitochondrial ROS production by flow cytometry (mean ± SE, n = 3 independent isolated platelets, two-way ANOVA with Sidak multiple comparisons test). c PAR3 and PAR4 expression in MTH1 fl/fl and MTH1 −/− platelets under resting conditions (mean ± SE, n = 3 independent isolated platelets, two-tailed unpaired Student’s t test). d Wild-type (WT) platelets were treated with thrombin (1 U/ml) or CRP (5 μg/ml) for 3 min followed by measuring the mitochondrial ROS generation using MitoSox Red and MitoTracker Green probes by fluorescent microscope (×100) ( n = 3 independent isolated platelets). e Representative image of mitochondrial ROS generation in WT platelets after stimulation with CRP or thrombin by flow cytometry. f WT platelets were pretreated with BAPTA (calcium inhibitor) (20 μM), BAY 11-7082 (NF-κB inhibitor), U-73122 (PLC inhibitor) (5 μM), LY294002 (PI3K inhibitor) (20 μM), PP1 (Src inhibitor) (10 μM) (MedChemExpress) for 5 min followed by stimulation with thrombin or CRP to measure mitochondrial ROS by flow cytometry (mean ± SE, n = 3 independent isolated platelets, one-way ANOVA with Dunnett multiple comparisons test). g WT platelets were pre-incubated with vehicle, Mito-TEMPO (10 μM) or Apocynin (500 μM) and then treated with thrombin or CRP followed by measuring intracellular ROS production by flow cytometry using H2DCFDA (mean ± SE, n = 3 independent isolated platelets, two-way ANOVA with Tukey multiple comparisons test).

Article Snippet: Human or wild-type mouse platelets were isolated to measure MTH1 expression by western blot with three different antibodies using human multiple myeloma (MM) cells or mouse acute myeloid leukemia (AML) cells as positive control (representative of three independent experiments) ( a ). b MTH1 expression in MTH1-deficient platelets was measured by western blot using MTH1 antibody (Santa Cruz). c Platelet cytosol and mitochondria were isolated from wild-type mice to measure the expression of MTH1 (MTH1 antibody from Santa Cruz), VDAC (VDAC antibody from Abcam) or GAPDH (representative of three independent experiments). d Platelets were isolated from wild-type mouse and then fixed followed by labeling with MTH1 antibody and then with the secondary antibody to evaluate the localization of MTH1 using immune electron microscopy ( n = 3 independent isolated platelets).

Techniques: Isolation, Flow Cytometry, Expressing, Two Tailed Test, Microscopy, Incubation

Journal: Nature Communications

Article Title: MTH1 protects platelet mitochondria from oxidative damage and regulates platelet function and thrombosis

doi: 10.1038/s41467-023-40600-7

Figure Lengend Snippet: a MTH1 fl/fl or MTH1 −/− platelets were treated with thrombin (1 U/ml) for 3 min followed by quantitative phosphoproteomics assay. b Differentially expressed phosphopeptides between two groups were presented as volcano map. X -axis shows the fold change (logarithmic conversion based on 2) and Y -axis shows the P-value (logarithmic conversion based on 10). Red dots represented the differentially upregulated phosphopeptides with significance and Blue dots showed the differentially downregulated phosphopeptides with significance. KEGG pathway analysis between control and MTH1-deficient platelets under the condition of resting (MA/NA) ( c ) or stimulation (MB/NB) ( d ). e MTH1 fl/fl or MTH1 −/− platelets were stimulated with thrombin (1 U/ml) followed by measuring the phosphorylation level of p38 MAPK, AKT, PLCβ3 and RhoA. The data were quantified based on three independent experiments (mean ± SD, n = 3 independent isolated platelets, two-way ANOVA with Sidak multiple comparisons test). f The number of differentially expressed phosphopeptides among the four groups. g Details of the 2 differentially expressed phosphopeptides localized in the mitochondria with significance identified from the comparison of control and MTH1-deficient platelets after thrombin stimulation ( n = 3 independent experiments, two-tailed unpaired Student’s t test).

Article Snippet: Human or wild-type mouse platelets were isolated to measure MTH1 expression by western blot with three different antibodies using human multiple myeloma (MM) cells or mouse acute myeloid leukemia (AML) cells as positive control (representative of three independent experiments) ( a ). b MTH1 expression in MTH1-deficient platelets was measured by western blot using MTH1 antibody (Santa Cruz). c Platelet cytosol and mitochondria were isolated from wild-type mice to measure the expression of MTH1 (MTH1 antibody from Santa Cruz), VDAC (VDAC antibody from Abcam) or GAPDH (representative of three independent experiments). d Platelets were isolated from wild-type mouse and then fixed followed by labeling with MTH1 antibody and then with the secondary antibody to evaluate the localization of MTH1 using immune electron microscopy ( n = 3 independent isolated platelets).

Techniques: Phospho-proteomics, Control, Isolation, Comparison, Two Tailed Test

Journal: Nature Communications

Article Title: MTH1 protects platelet mitochondria from oxidative damage and regulates platelet function and thrombosis

doi: 10.1038/s41467-023-40600-7

Figure Lengend Snippet: a Western blot analysis of the expression of MUTYH, OGG1/2, MTH2, and MTH3 in MTH1 fl/fl and MTH1 -/- platelets under resting condition. The representative images were shown from three independent experiments (mean ± SD, n = 3 independent isolated platelets). b Mice were administered a Dylight 488-labeled anti-GP1bβ antibody (Emfret, X488) via tail vein (0.1 μg/g body weight) to measure platelet half-life by flow cytometry. Data were presented as mean ± SE ( n = 3, two-way ANOVA with Sidak multiple comparisons test. c Expression of subunit of complexes (CI, CIII, CIV and CV) in the mitochondrial respiration chain proteins encoded by mtDNA (ND1, CYTB, MT-CO1, ATP8) or nucleus DNA (NDUFV1, UQCRC2, COX6A1, ATP5A) in platelets from MTH1 fl/fl or MTH1 −/− mice before (−) and after (+) stimulation with thrombin (1 U/ml) for 3 mins (representative of three independent experiments) (mean ± SD, n = 3, two-way ANOVA with Sidak multiple comparisons test). d MT-CO1 gene expression level in thrombin-activated platelets from MTH1 fl/fl or MTH1 −/− mice was measured by quantitative real-time PCR and represented as a fold change relative to its level in resting platelets (mean ± SD, n = 3 independent isolated platelets, two-tailed unpaired Student’s t test). e Analysis of the number of guanine in the 13 mtDNA sequences.

Article Snippet: Human or wild-type mouse platelets were isolated to measure MTH1 expression by western blot with three different antibodies using human multiple myeloma (MM) cells or mouse acute myeloid leukemia (AML) cells as positive control (representative of three independent experiments) ( a ). b MTH1 expression in MTH1-deficient platelets was measured by western blot using MTH1 antibody (Santa Cruz). c Platelet cytosol and mitochondria were isolated from wild-type mice to measure the expression of MTH1 (MTH1 antibody from Santa Cruz), VDAC (VDAC antibody from Abcam) or GAPDH (representative of three independent experiments). d Platelets were isolated from wild-type mouse and then fixed followed by labeling with MTH1 antibody and then with the secondary antibody to evaluate the localization of MTH1 using immune electron microscopy ( n = 3 independent isolated platelets).

Techniques: Western Blot, Expressing, Isolation, Labeling, Flow Cytometry, Gene Expression, Real-time Polymerase Chain Reaction, Two Tailed Test

Journal: Nature Communications

Article Title: MTH1 protects platelet mitochondria from oxidative damage and regulates platelet function and thrombosis

doi: 10.1038/s41467-023-40600-7

Figure Lengend Snippet: Washed human platelets were pre-treated with 5 μM TH588 (MTH1 inhibitor) for 1 h at 37 °C and platelet aggregation and ATP release induced by either thrombin (0.04 U/ml) (mean ± SD, n = 3, two-tailed unpaired Student’s t test) ( a ) or CRP (0.5 μg/ml) (mean ± SD, n = 3, two-tailed unpaired Student’s t test) ( b ), as well as 8-oxo-dG accumulation in platelet mitochondria induced by thrombin (1 U/ml) or CRP (5 μg/ml) ( c ) (mean ± SD, n = 3, two-way ANOVA with Sidak multiple comparisons test) were measured.

Article Snippet: Human or wild-type mouse platelets were isolated to measure MTH1 expression by western blot with three different antibodies using human multiple myeloma (MM) cells or mouse acute myeloid leukemia (AML) cells as positive control (representative of three independent experiments) ( a ). b MTH1 expression in MTH1-deficient platelets was measured by western blot using MTH1 antibody (Santa Cruz). c Platelet cytosol and mitochondria were isolated from wild-type mice to measure the expression of MTH1 (MTH1 antibody from Santa Cruz), VDAC (VDAC antibody from Abcam) or GAPDH (representative of three independent experiments). d Platelets were isolated from wild-type mouse and then fixed followed by labeling with MTH1 antibody and then with the secondary antibody to evaluate the localization of MTH1 using immune electron microscopy ( n = 3 independent isolated platelets).

Techniques: Two Tailed Test

Journal: Journal of Extracellular Vesicles

Article Title: Impact on NK cell functions of acute versus chronic exposure to extracellular vesicle‐associated MICA: Dual role in cancer immunosurveillance

doi: 10.1002/jev2.12176

Figure Lengend Snippet: EV‐associated MICA*008 and not its soluble form induces NKG2D downmodulation. (a) Conditioned supernatants derived from ARK MICA*008 transfectants or ARK transfected with an empty vector were depleted of EVs and used to culture the NKL cell line for 24 h. NKG2D and DNAM‐1 expression was evaluated by immunofluorescence and FACS analysis. NKG2D relative expression was calculated respect to the untreated group (= 100%) represented by dashed line. Values reported represent the mean of independent experiments. (b‐f) ARK cell transfectants were cultured for 48 h in EV‐free medium and then EVs were isolated from the conditioned media as described in materials and methods. (b) Size distribution of sEVs and mEVs was analysed through dynamic light scattering (DLS). A representative experiment is shown. (c) Transmission electron microscopy (TEM) of sEVs and mEVs preparations. Bar corresponds to 200 nm. (d) The amount of total proteins recovered from EV preparations derived from 10 6 cells was calculated by BPA and corresponded to 1.127 μg ± 0.58 for sEVs and 3.04 μg ± 1.79 for mEVs. (e) The number of EVs/μg of protein was calculated through DLS. (f) Western blot analysis was performed on lysates (40 μg) derived from sEVs and mEVs fractions or from cell pellet, using anti‐Hsp70, anti‐calreticulin, anti‐CD63, anti‐MHC I and anti‐CD81 antibodies

Article Snippet: Real‐time PCR was performed using the ABI Prism 7900 Sequence Detection system (Applied Biosystems, Foster City, CA). cDNAs were amplified in triplicate using the following specific TaqMan Gene Expression Assay all conjugated with fluorochrome FAM (Applied Biosystems): IFNγ (Hs00989291_m1), MICA (Hs00792195_m1) and human β‐actin (Hs99999903_m1).

Techniques: Derivative Assay, Transfection, Plasmid Preparation, Expressing, Immunofluorescence, Cell Culture, Isolation, Transmission Assay, Electron Microscopy, Western Blot

Journal: Journal of Extracellular Vesicles

Article Title: Impact on NK cell functions of acute versus chronic exposure to extracellular vesicle‐associated MICA: Dual role in cancer immunosurveillance

doi: 10.1002/jev2.12176

Figure Lengend Snippet: Characterization and functional activity of sEVs and mEVs expressing MICA*008. (a) Western blot analysis was performed on lysates derived from sEVs and mEVs fractions or from cell pellet of ARK transfectants, using anti‐MICA or anti‐MHC I antibodies. (b) Lysates derived fromsEVs and mEVs were assessed for the presence of MICA using a specific ELISA. Values are represented as the amount of MICA/μg of proteins and represent the mean of four independent experiments. (c) Immunofluorescence and FACS analysis of mEVs or CD63 + beads coated with sEVs and labelled with control isotypic Ig (cIg), anti‐CD63 or anti‐MICA Abs, as indicated in the figure. A representative experiment is shown. (d) Graph is referred to the mean of different experiments and values represent the mean fluorescence intensity (MFI) of MICA subtracted from the MFI value of the cIg. (e and f) The NKL cell line was incubated for 24 h with 30 μg/ml of sEVs or 20 μg/ml of mEVs; cells were collected and NKG2D expression was evaluated by immunofluorescence and FACS analysis. A representative experiment is shown. In (f) values reported represent the mean of different independent experiments. Relative expression of NKG2D was calculated considering the untreated group as 100% (dashed line)

Article Snippet: Real‐time PCR was performed using the ABI Prism 7900 Sequence Detection system (Applied Biosystems, Foster City, CA). cDNAs were amplified in triplicate using the following specific TaqMan Gene Expression Assay all conjugated with fluorochrome FAM (Applied Biosystems): IFNγ (Hs00989291_m1), MICA (Hs00792195_m1) and human β‐actin (Hs99999903_m1).

Techniques: Functional Assay, Activity Assay, Expressing, Western Blot, Derivative Assay, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Control, Fluorescence, Incubation

Journal: Journal of Extracellular Vesicles

Article Title: Impact on NK cell functions of acute versus chronic exposure to extracellular vesicle‐associated MICA: Dual role in cancer immunosurveillance

doi: 10.1002/jev2.12176

Figure Lengend Snippet: MICA*008 EVs are taken up more efficiently by NK cells. The NKL cell line was incubated for 3 h with 30 μg/ml of PKH26‐labelled sEVs or 20 μg/ml of PKH26‐labelled mEVs derived from ARK transfectants. The fluorescence of internalized EVs was evaluated by FACS analysis and measured as the percentage of PKH26 + cells. (a) One representative experiment is shown. (b) The mean of six independent experiments is shown. (c) NKL cells were pre‐treated for 1 h with EDTA (10 mM), EIPA (50 μM), dynasore (50 μM), nystatin (40 μg/ml) and then incubated for 3 h with PKH26‐labelled EVs. The fluorescence of internalized EVs was evaluated by FACS and measured as the percentage of PKH26 positive cells referred to untreated cells which were considered as 100%. Values reported represent the mean of at least three independent experiments

Article Snippet: Real‐time PCR was performed using the ABI Prism 7900 Sequence Detection system (Applied Biosystems, Foster City, CA). cDNAs were amplified in triplicate using the following specific TaqMan Gene Expression Assay all conjugated with fluorochrome FAM (Applied Biosystems): IFNγ (Hs00989291_m1), MICA (Hs00792195_m1) and human β‐actin (Hs99999903_m1).

Techniques: Incubation, Derivative Assay, Fluorescence

Journal: Journal of Extracellular Vesicles

Article Title: Impact on NK cell functions of acute versus chronic exposure to extracellular vesicle‐associated MICA: Dual role in cancer immunosurveillance

doi: 10.1002/jev2.12176

Figure Lengend Snippet: NKG2D mediates the uptake of MICA*008 + EVs. (a) NKL cells were pre‐treated with αNKG2D and αDNAM‐1 blocking mAbs and then incubated for 3 h with PKH26‐labelled sEVs (30 μg/ml) or mEVs (20 μg/ml). Data reported represent the mean of three independent experiments. Values represent the percentage of PKH26 + cells. (b and c) NKL cells were treated overnight with MICA*008 + EVs to induce NKG2D downmodulation (NKG2D low ) or left untreated (NKG2D high ). (b) A representative experiment showing the different levels of NKG2D expression is shown. (c) The mean of three independent experiments is shown. (d) NKG2D low and NKG2D high NKL cells were incubated with PKH26‐labelled EVs for 3 h as described in panel (a). Values represent the percentage of PKH26 + cells. The mean of three independent experiments is shown. (e–g) NKL cells were infected with the pLKO lentiviral vector containing a scrambled sequence or shRNA for silencing NKG2D. NKG2D expression was evaluated by (e) FACS analysis and (f) real‐time PCR. (g) NKL/pLKO and NKL/shNKG2D were incubated with PKH26‐labelled EVs for 3 h as described in panel (a). Values represent the percentage of PKH26 + cells. The mean of at least four independent experiments is shown. (h) Ba/F3 or Ba/F3 stably expressing the NKG2D/DAP10 complex were stained with cIg or anti‐NKG2D mAb and analyzed by Immunofluorescence and FACS analysis. (i) Ba/F3 or Ba/F3‐NKG2D/DAP10 were plated at 5 × 10 5 cells/ml and incubated for 1 h with PKH26‐labelled mEVs as described in panel (a). Values represent the percentage of PKH26 + cells. The mean of three (for sEVs) or four (for mEVs) independent experiments is shown

Article Snippet: Real‐time PCR was performed using the ABI Prism 7900 Sequence Detection system (Applied Biosystems, Foster City, CA). cDNAs were amplified in triplicate using the following specific TaqMan Gene Expression Assay all conjugated with fluorochrome FAM (Applied Biosystems): IFNγ (Hs00989291_m1), MICA (Hs00792195_m1) and human β‐actin (Hs99999903_m1).

Techniques: Blocking Assay, Incubation, Expressing, Infection, Plasmid Preparation, Sequencing, shRNA, Real-time Polymerase Chain Reaction, Stable Transfection, Staining, Immunofluorescence

Journal: Journal of Extracellular Vesicles

Article Title: Impact on NK cell functions of acute versus chronic exposure to extracellular vesicle‐associated MICA: Dual role in cancer immunosurveillance

doi: 10.1002/jev2.12176

Figure Lengend Snippet: MICA*008 + EVs are preferentially uptaken by leukocyte subpopulations expressing NKG2D. Human primary peripheral blood mononuclear cells (PBMC) were incubated for 3 h with PKH26‐labelled mEVs expressing or not MICA*008. (a) Representative dot plot of different PBMC populations, by gating on CD45 + CD56 + CD3 – for NK cells, on CD4 + and CD8 + after gating CD45 + CD56 – CD3 + for T lymphocytes. Representative uptake of these populations is shown. Values reported in each plot represent the percentage of PKH26 + cells. (b) The mean of at least four independent experiments is shown. (c) FACS analysis of NKG2D expression on different leukocyte populations. A representative plot is shown

Article Snippet: Real‐time PCR was performed using the ABI Prism 7900 Sequence Detection system (Applied Biosystems, Foster City, CA). cDNAs were amplified in triplicate using the following specific TaqMan Gene Expression Assay all conjugated with fluorochrome FAM (Applied Biosystems): IFNγ (Hs00989291_m1), MICA (Hs00792195_m1) and human β‐actin (Hs99999903_m1).

Techniques: Expressing, Incubation

Journal: Journal of Extracellular Vesicles

Article Title: Impact on NK cell functions of acute versus chronic exposure to extracellular vesicle‐associated MICA: Dual role in cancer immunosurveillance

doi: 10.1002/jev2.12176

Figure Lengend Snippet: MICA*008 positive EVs engage NKG2D and trigger pERK and IFN‐γ production. (a) NKL cells were treated for different times with MICA*008 + or MICA*008 – sEVs (at 30 μg/ml) or mEVs (at 20 μg/ml). Cells were harvested and NKG2D expression was evaluated by immunofluorescence and FACS analysis. Relative expression of NKG2D was calculated considering the untreated group as 100%. (b) NKL cells were treated for 1 h with MICA*008 + or MICA*008 – EVs. Western blot analysis was performed on total cell lysates using ERK and phospho‐ERK (pERK) Abs. Numbers beneath each lane represent quantification of pERK by densitometry analysis normalized with ERK relative to the untreated group. (c) Quantification of immunoblots relative to different independent experiments is shown. Dashed line represents the untreated group. (d) Highly purified human primary NK cells were incubated with 30 μg/ml of sEVs or 20 μg/ml of mEVs expressing or not MICA*008 for 24 h. Real‐time PCR analysis of IFN‐γ mRNA is shown. Data, expressed as fold change units, were normalized with β‐actin and referred to MICA*008 – EVs treated cells considered as calibrator. Values reported represent the mean of at least three independent experiments

Article Snippet: Real‐time PCR was performed using the ABI Prism 7900 Sequence Detection system (Applied Biosystems, Foster City, CA). cDNAs were amplified in triplicate using the following specific TaqMan Gene Expression Assay all conjugated with fluorochrome FAM (Applied Biosystems): IFNγ (Hs00989291_m1), MICA (Hs00792195_m1) and human β‐actin (Hs99999903_m1).

Techniques: Expressing, Immunofluorescence, Western Blot, Purification, Incubation, Real-time Polymerase Chain Reaction

Journal: Journal of Extracellular Vesicles

Article Title: Impact on NK cell functions of acute versus chronic exposure to extracellular vesicle‐associated MICA: Dual role in cancer immunosurveillance

doi: 10.1002/jev2.12176

Figure Lengend Snippet: Prolonged exposure to MICA*008 + EVs impairs both NKG2D signalling pathway and NKG2D‐mediated killing. NKL cells were treated for 24 h with 30 μg/ml of sEVs or 20 μg/ml of mEVs expressing or not MICA*008. (a) Untreated or EV‐treated NKL cells were stimulated with anti‐NKG2D mAb plus goat anti‐mouse IgG for 15 min at 37°C before the lysis. Western blot analysis was performed on total cell lysates using ERK and phospho‐ERK (pERK) Abs. Numbers beneath each lane represent quantification of pERK by densitometry analysis normalized with ERK relative to the untreated group. (b) Quantification of immunoblots relative to independent experiments is shown. (c) The cytotoxic activity of EV‐treated NKL cells was evaluated by performing the 7‐AAD assay in a redirected modality against P815 FcR+ target cells loaded with different Ab as indicated in figure, at an E:T ratio of 25:1. Data reported represent the mean of at least three independent experiments. d) The NKL intracellular amount of perforin, granzyme A, B and K was evaluated through immunofluorescence and FACS analysis. The mean of at least three independent experiments is shown. Values represent the MFI of granzyme A, B, K and perforin subtracted from the MFI value of the cIg

Article Snippet: Real‐time PCR was performed using the ABI Prism 7900 Sequence Detection system (Applied Biosystems, Foster City, CA). cDNAs were amplified in triplicate using the following specific TaqMan Gene Expression Assay all conjugated with fluorochrome FAM (Applied Biosystems): IFNγ (Hs00989291_m1), MICA (Hs00792195_m1) and human β‐actin (Hs99999903_m1).

Techniques: Expressing, Lysis, Western Blot, Activity Assay, Immunofluorescence

Journal: Journal of Extracellular Vesicles

Article Title: Impact on NK cell functions of acute versus chronic exposure to extracellular vesicle‐associated MICA: Dual role in cancer immunosurveillance

doi: 10.1002/jev2.12176

Figure Lengend Snippet: MICA*008 + mEVs induce NKG2D downmodulation ‘in vivo’. (a) Purified spleen‐derived mouse NK cells were plated at 2 × 10 6 cells/ml and treated with mEVs (20 μg/ml) for 3 and 18 h in the presence of IL‐15 (10 ng/ml). NKG2D expression levels were evaluated by immunofluorescence and FACS analysis. A representative experiment out of two is shown. (b) NKG2D downmodulation in vivo was assessed after i.p. injection of mEVs (50 μg) into mice. After 3 and 18 h, PB and peritoneal cells were stained with anti‐CD45.2, anti‐NK1.1, anti‐CD3 and anti‐NKG2D mAbs. NKG2D expression was measured by gating on NK.1.1 + CD3 – CD45 + cells. Results represent average of two independent experiments +/‐ SEM performed. (c) Host mice were i.p. treated with mEVs expressing or not MICA*008 (50 μg) 18 h before and at the time of target cell injection. Mice were i.p. injected with equal numbers (10 6 ) of target (5TGM1) and control (RMA) cells labelled with CFSE and PKH26, respectively. Cells were harvested from the PC 4 h later, stained with annexin V and the ratio of surviving target to control cells was calculated as number of annexin V – CFSE + PKH 26 + cells. Values represent average obtained from two independent experiments +/‐ SEM performed. d) In order to deplete NK1.1 + cells, anti‐NK1.1 mAb (Pk136, 100 μg/mouse) was administered to mice by i.p. injection the day before the assay. A total of 1 × 10 6 CFSE‐labelled live 5TGM1 cells were injected i.p. into C57BL/6 NK cell competent or NK cell depleted mice. Four hours later, mice were sacrificed and a peritoneal lavage performed. The percentage (number in gate) of CFSE‐labelled cells within the peritoneal lavage was determined by flow cytometry. One representative analysis is shown

Article Snippet: Real‐time PCR was performed using the ABI Prism 7900 Sequence Detection system (Applied Biosystems, Foster City, CA). cDNAs were amplified in triplicate using the following specific TaqMan Gene Expression Assay all conjugated with fluorochrome FAM (Applied Biosystems): IFNγ (Hs00989291_m1), MICA (Hs00792195_m1) and human β‐actin (Hs99999903_m1).

Techniques: In Vivo, Purification, Derivative Assay, Expressing, Immunofluorescence, Injection, Staining, Control, Flow Cytometry

Journal: Journal of Extracellular Vesicles

Article Title: Impact on NK cell functions of acute versus chronic exposure to extracellular vesicle‐associated MICA: Dual role in cancer immunosurveillance

doi: 10.1002/jev2.12176

Figure Lengend Snippet: MICA*008 is transferred by EVs on NK cells. (a and b) NKL cells were incubated for different times with 30 μg/ml of sEVs and 20 μg/ml of mEVs expressing or not MICA*008 and after that MICA expression was evaluated by immunofluorescence and FACS analysis. In (a) data relative to a representative experiment are shown. Numbers above histograms represent the percentage of MICA + cells. In (b) the mean of different independent experiments is shown. (c) NKL cells were treated as described in panel (a) and (b). Cells were harvested at different times as indicated, and real‐time PCR of MICA mRNA was performed. Data, expressed as fold change, were normalized with β‐actin and referred to MICA*008 – EVs treated cells considered as calibrator (dashed line). Values reported represent the mean of at least three independent experiments. (d) NKL cells were pre‐treated with anti‐NKG2D and anti‐DNAM‐1 blocking Abs and then incubated for 3 h with 30 μg/ml of sEVs and 20 μg/ml of mEVs expressing or not MICA*008. MICA expression on NKL cells was evaluated by immunofluorescence and FACS analysis. Data reported represent the mean of four independent experiments. (e–g) NKL cells were labelled with CFSE and then cocultured with NKL cells dressed with MICA for 18 h and NKG2D expression was evaluated on CFSE + NKL cells. A representative experiment is shown in panel (e). In (f) NKG2D MFI values relative to six independent experiments are shown. In (g) data are expressed as relative expression of NKG2D (MFI) respect to the untreated group (CFSE + NKL cells corresponded to 100%)

Article Snippet: Real‐time PCR was performed using the ABI Prism 7900 Sequence Detection system (Applied Biosystems, Foster City, CA). cDNAs were amplified in triplicate using the following specific TaqMan Gene Expression Assay all conjugated with fluorochrome FAM (Applied Biosystems): IFNγ (Hs00989291_m1), MICA (Hs00792195_m1) and human β‐actin (Hs99999903_m1).

Techniques: Incubation, Expressing, Immunofluorescence, Real-time Polymerase Chain Reaction, Blocking Assay

Journal: Journal of Extracellular Vesicles

Article Title: Impact on NK cell functions of acute versus chronic exposure to extracellular vesicle‐associated MICA: Dual role in cancer immunosurveillance

doi: 10.1002/jev2.12176

Figure Lengend Snippet: Dressing of NK cells with MICA*008 by EVs induces fratricide. (a) Human polyclonal NK cells were treated for 18 h with 30 μg/ml of sEVs and 20 μg/ml of mEVs expressing or not MICA*008. MICA expression on NK cells was evaluated by immunofluorescence and FACS analysis. Values reported represent the percentage of MICA + cells and represent the mean of different independent experiments. (b and c) Polyclonal NK cells were labelled with CFSE and used as effectors in a CD107 degranulation assay. As target cells autologous CFSE – polyclonal NK cells treated or not with EVs were used (E:T ratio: 2.5:1). A representative experiment is shown. Values in each plot indicate the percentage of CD107 + CFSE + NK cells. In (c) data relative to five independent experiments are shown. (d) Polyclonal NK cells were labelled with CFSE and used as effectors in a 7AAD cytotoxicity assay. As target cells autologous CFSE – polyclonal NK cells treated or not with EVs were used (E:T ratio: 12.5:1). The percentage of CFSE – 7AAD + NK cells was evaluated by immununofluorescence and FACS analysis. The mean of three independent experiments is shown

Article Snippet: Real‐time PCR was performed using the ABI Prism 7900 Sequence Detection system (Applied Biosystems, Foster City, CA). cDNAs were amplified in triplicate using the following specific TaqMan Gene Expression Assay all conjugated with fluorochrome FAM (Applied Biosystems): IFNγ (Hs00989291_m1), MICA (Hs00792195_m1) and human β‐actin (Hs99999903_m1).

Techniques: Expressing, Immunofluorescence, Degranulation Assay, Cytotoxicity Assay

Journal: Journal of Extracellular Vesicles

Article Title: Impact on NK cell functions of acute versus chronic exposure to extracellular vesicle‐associated MICA: Dual role in cancer immunosurveillance

doi: 10.1002/jev2.12176

Figure Lengend Snippet: MICA is associated to BM‐derived EVs of MM patients. mEVs were isolated from peripheral blood (PB) or bone marrow (BM) aspirates of a cohort of MM patients. (a) Ultrastructural analysis of a representative sample. Bar corresponds to 200 nm. (b) Immunofluorescence and FACS analysis of mEVs using antibodies against MHC I, CD138 and MICA the expression of which is reported by percentage

Article Snippet: Real‐time PCR was performed using the ABI Prism 7900 Sequence Detection system (Applied Biosystems, Foster City, CA). cDNAs were amplified in triplicate using the following specific TaqMan Gene Expression Assay all conjugated with fluorochrome FAM (Applied Biosystems): IFNγ (Hs00989291_m1), MICA (Hs00792195_m1) and human β‐actin (Hs99999903_m1).

Techniques: Derivative Assay, Isolation, Immunofluorescence, Expressing