Journal: Cancers

Article Title: Molecular Interplay between Dormant Bone Marrow-Resident Cells (BMRCs) and CTCs in Breast Cancer

doi: 10.3390/cancers12061626

Figure Lengend Snippet: Validation of ex vivo BMRC/CTC using complementary imaging platforms. ( A ) Detection of ex vivo human CTCs by CellSearch CTC (epithelial) kit. The kit enumerates CTCs using DAPI, Pan-cytokeratin-PE (CK-PE), and CD45-APC staining. Upper panel shows a CTC clustering with a CD45+ cell (lymphocyte); lower panels show an individual CTC. Image resolution achieved in accordance with the USFDA-approved CellSearch platform. ( B ) Immunofluorescent (IF) staining of ex vivo BMRC using GCDFP15/MG cocktail. Image of a BMRC showing red IF staining against mouse primary antibody clones 23A3 (GCDFP-15) and 304-1A5 (MG), and green IF staining for rabbit clone 31A5 (MG). ( C ) IF images of single ex vivo epithelial and stem-like BMRCs and CTCs. ( D ) DEPArray images of ex vivo epithelial and stem-like BMRCs and CTCs. Image magnification: 10×. The DEPArray platform allows the capture, visualization, and downstream interrogation of single CTCs. Since the image resolution and magnification obtained from FDA-cleared CellSearch and DEPArray platforms cannot be altered by the user, we used IF to provide a high-resolution image of captured ex vivo BMRC/CTCs.

Article Snippet: IHC on mouse tissue using antibodies of mouse origin were performed using M.O.M. elite peroxidase kit; dual IHCs were performed using ImmPRESS Duet Double Staining HRP/AP Polymer Kit, and triple IHCs were performed by multiplexing with ImmPRESS-AP Anti-Rat IgG, Mouse Adsorbed Polymer Detection Kit (Vector Labs, Burlingame, CA, USA).

Techniques: Ex Vivo, Imaging, Staining, Clone Assay

Journal: Journal of translational medicine

Article Title: Unraveling resistance mechanisms in anti-CD19 chimeric antigen receptor-T therapy for B-ALL: a novel in vitro model and insights into target antigen dynamics.

doi: 10.1186/s12967-024-05254-z

Figure Lengend Snippet: Fig. 1 Proliferation and specific cytotoxic effects of CART-19 cells. A The design of the CAR-T cell construction experiments. B Morphological images of activated T cells clustered after 24 h and 72 h of incubation with TransAct CD3/28 beads. C Flow cytometric analysis of CAR expression on the surface of mock T, and CART-19 cells with biotin-conjugated anti-Fab antibody followed by PE-conjugated streptavidin. Gating was based on the same cells stained with isotype-matched antibody. The median fluorescence intensity (MFI) was calculated for CAR-T population in the PE fluorescence channel (right column). This result is the representative of three separate experiments using cells from healthy volunteer donors. D The phenotypic characterization of CART-19 cells by flow cytometry. The ratio of CD4+ / CD8+ T cells (left) and the proportion of TN/CM (right) are shown. E Growth curves of CAR-T cells. Data represent the mean ± s.d. of three separate experiments. F Cytolytic activities of CART-19 cells in cell assays. Nalm-6 cells were labeled with CFSE labeling reagent (Sigma-Aldrich, USA) and co-cultured with CART-19 cells at the E: T ratio of 1:1 for 30 h. The presence of CFSE-labeled cells was observed by mi croscopy. Bar, 100 μm. G Cytotoxic activity of mock NT and CART cells against Nalm-6 cells. The effector cells were co-cultured with target cells at E: T ratios of 1:5, 1:2, 1:1 and 5:1 with a total cell number of 1 × 106. H Dynamic changes of cytokine secretion profile of CART-19 cells during 24 h after co-culture with Nalm-6 cells at E: T ratios of 1:5 to 5:1. Data were visualized by heatmap. Concentrations (pg/ml) of cytokines and chemokines in the supernatant were detected by multiplex immunoassay and the values were log2 transformed

Article Snippet: To evaluate CAR expression after 7–10 days of culture, CART-19 cells were washed once and incubated with goat anti-human biotin conjugated anti-Fab antibody (Jackson ImmunoResearch, USA) for 30 min at room temperature.

Techniques: Incubation, Expressing, Staining, Fluorescence, Flow Cytometry, Labeling, Cell Culture, Activity Assay, Co-Culture Assay, Multiplex Assay, Transformation Assay

Journal: Journal of translational medicine

Article Title: Unraveling resistance mechanisms in anti-CD19 chimeric antigen receptor-T therapy for B-ALL: a novel in vitro model and insights into target antigen dynamics.

doi: 10.1186/s12967-024-05254-z

Figure Lengend Snippet: Fig. 5 Observation of CD19-BBζ-CAR expression in relapsed Nalm-6 cells and salvage treatment. A Detection of FMC63 and CD247 transcripts and 4-1BB gene of CAR in CD19+ Nalm-6 (red) and relapsed CD19− Nalm-6 cells (blue) by qRT-PCR. Data of left bar graph represent the relative quantification using ACTB as the internal reference. Error bars represent s.d. The data are the representative of three independent experiments. B Expression of CD19 and CAR on CD19+ Nalm-6 cells and relapsed CD19− Nalm-6 cells analyzed by flow cytometry (representative of 3 experiments). Merge Graphs, the blue dots represent CD19− Nalm-6 cells and the red dots represent Nalm-6 cells. C Confocal imaging of Nalm-6 cells and relapsed CD19− Nalm-6 cells using Alexa Flour 488-conjugated anti-CD19 antibody (green), Alexa Flour 647-conjugated anti-CAR19 antibody (red), and DAPI (blue). D Lentiviral integration sites of CAR transduced Nalm-6 cells were analyzed by linear-amplification mediated PCR (LAM-PCR) and visualized with Circos plots. The integration sites across the genome and genomic features were shown from outer to inner circle: (1) cytogenetic bands; (2) genes that harbor these integration sites along with a bar chart showing the reads of integration sites; (3) the distribution of integration sites, with colored circles representing different gene functional regions of the host sequence: purple for promoter region, green for intron region, and red for distal intergenic region. E Phenotype changes of Nalm-6 cells transduced with small amount of CD19 CAR lentiviruses detected by flow cytometry over time. Gating was based on the same cells stained with isotype-matched antibody. F Dynamics of CD19− B phenotype in relapsed cells after co-culture with different ratios (5×, 20×) of Nalm-6 cells. Gating was based on the same cells stained with isotype-matched antibody. G Relapsed CD19− Nalm-6 cells were tested by qPCR specific for VSV-G sequence. H Comparison of in vitro efficacy of CD19-, CD22-, CD19/CD22- and CD22×CD19- CAR T cells. Cocultures with the relapsed cells were performed at 1:5, 1:1, and 5:1 E: T ratios, and lysis efficacies were detected by the LDH release assay Declarations

Article Snippet: To evaluate CAR expression after 7–10 days of culture, CART-19 cells were washed once and incubated with goat anti-human biotin conjugated anti-Fab antibody (Jackson ImmunoResearch, USA) for 30 min at room temperature.

Techniques: Expressing, Quantitative RT-PCR, Quantitative Proteomics, Flow Cytometry, Imaging, Amplification, Functional Assay, Sequencing, Transduction, Staining, Co-Culture Assay, Comparison, In Vitro, Lysis, Lactate Dehydrogenase Assay

Journal: bioRxiv

Article Title: AAV gene therapy for GBA-PD and Gaucher Disease

doi: 10.1101/2025.06.17.660133

Figure Lengend Snippet: (A) A schematic depicting our strategy to generate GBA1 variants with enhanced secretion. Endogenous signal sequence of human GBA1 was swapped with signal sequences from highly secreted proteins. Top 4 signal sequences (SS) were narrowed down using in-silico tools that predicted robust secretion as well as high (>96%) probability of cleavage at the end of signal sequence. (B) HEK293T cells were transfected with GBA1 plasmid containing the indicated SS variants, lysed, and GCase enzyme activity was determined in cell lysates. Mean ± SEM, n = 4 independent experiments. Untransfected versus all GBA1 constructs as well as GFP transfected versus all GBA1 constructs: ***p<0.001; One-way ANOVA with Turkey’s multiple comparison test. (C) Representative immunofluorescence images demonstrating co-localization of active GCase and lysosomes. HEK293T cells were transfected with GBA1 variant constructs, incubated with MDW933 fluorescence probe to label active GCase, and immunostained with Lamp1 antibody for lysosomes. Blue and yellow arrowheads point to individual puncta showing co-localization of GCase with Lamp1. Scale bar is 10 microns. (D and E) HEK293T cells were transfected with GBA1 variant constructs and cell culture media was collected to detect secreted GCase (D) and measure GCase enzyme activity (E). Mean ± SEM, n = 4 independent experiments. Untransfected versus all GBA1 constructs as well as GFP transfected versus all GBA1 constructs: ***p<0.001; One way-ANOVA with Tukey’s multiple comparisons test.

Article Snippet: The slides were then incubated with human GBA1 antibodies (1:100, abcam, ab125065 and 1:400, Novus, NBP2-45829) for rodent and NHP, respectively.

Techniques: Sequencing, In Silico, Transfection, Plasmid Preparation, Activity Assay, Construct, Comparison, Immunofluorescence, Variant Assay, Incubation, Fluorescence, Cell Culture

Journal: bioRxiv

Article Title: AAV gene therapy for GBA-PD and Gaucher Disease

doi: 10.1101/2025.06.17.660133

Figure Lengend Snippet: (A) Study design: AAV GMU01 capsid expressing the indicated GBA1 variants were administered by bilateral ICV to 4 month-old C57/BL6 mice at 1e11 vector genomes per animal and 5 μl per hemisphere. N=4 animals per group. The sagittal sections of brain hemisphere were analyzed 4 weeks post-injection. (B) Representative images demonstrating GCase secretion with engineered variants. Vector biodistribution is shown with in situ hybridization to WPRE (top panels) and GCase is shown with immunohistochemistry. (C) Representative images demonstrating cross-correction. Higher magnification images of WPRE mRNA (left) and GCase protein (right) in SS3-GBA1 injected mice brain, image corresponds to the purple box in (A). Cells positive for both WPRE mRNA and GCase are shown in red arrows (AAV-transduced cells) while mRNA negative and GCase positive cells are shown in green arrows (cross-corrected cells). Scale bar is 500 microns.

Article Snippet: The slides were then incubated with human GBA1 antibodies (1:100, abcam, ab125065 and 1:400, Novus, NBP2-45829) for rodent and NHP, respectively.

Techniques: Expressing, Plasmid Preparation, Injection, In Situ Hybridization, Immunohistochemistry

Journal: bioRxiv

Article Title: AAV gene therapy for GBA-PD and Gaucher Disease

doi: 10.1101/2025.06.17.660133

Figure Lengend Snippet: (A) Measurement of Lyso-GL1 lipid substrate levels by LC-MS in cortex and liver of C57/BL6 mice 24 hours post CBE injection by IP injection at indicated doses. N=7 animals for CBE 0 mg/kg and N=4 per all the other groups. Data are Mean ± SEM. (B) Kinetic analysis of Lyso-GL1 lipid substrate by LC-MS in cortex and liver of 8-month-old mice from three different genotypes ( Gba D409V/D409V , Gba D409V/+, and Gba +/+ ). Mice with 100 mg/kg CBE IP dosing. Data are Mean ± SEM. 8 – 10 animals at each time point. (C) Study design: AAV GMU01 capsid expressing the indicated GBA1 variants were administered by bilateral ICV to 4-month-old C57/BL6 mice at 1e11 vector genomes per animal and 5μl per hemisphere. N=8 animals per group. CBE (conduritol β-epoxide) was administered via IP injection at 100 mg/kg 24 hours prior to necropsy. (D) Representative image of in situ hybridization to WPRE mRNA from the analyzed sagittal sections. Cortex is proximal to site of injection and cerebellum is distal to site of injection. (E-G) Measurement of Lyso-GL1 lipid substrate levels by LC-MS in midbrain (E), cerebellum (F), and hindbrain (G). No CBE group in the graphs shown is control mice that did not receive any AAV vector or CBE injection. 8 mice per group. ***p<0.001, **p<0.01, *p<0.05; One way- ANOVA with Tukey’s multiple comparisons test with all groups compared to CBE-treated vehicle injected group.

Article Snippet: The slides were then incubated with human GBA1 antibodies (1:100, abcam, ab125065 and 1:400, Novus, NBP2-45829) for rodent and NHP, respectively.

Techniques: Liquid Chromatography with Mass Spectroscopy, Injection, Expressing, Plasmid Preparation, In Situ Hybridization, Control

Journal: bioRxiv

Article Title: AAV gene therapy for GBA-PD and Gaucher Disease

doi: 10.1101/2025.06.17.660133

Figure Lengend Snippet: (A) Study design: 2-3 years old cynomolgus monkeys (2-3kg) were dosed either with AAV GMU01-WT-GBA1 or AAV GMU01-SS3-GBA1 at 1.25e13 vector genomes per animal by direct injection to the cisterna magna (ICM) in 2.5 ml volume at 0.125 ml/min rate. 6 weeks post- dosing, 30 mg/kg of CBE was administered by IV injection 48 hours prior to necropsy. Samples represent 64 brain biopsy punches encompassing 19 distinct grey matter regions and 7 distinct white matter regions. (B and C) AAV vector genome copies determined from 64 brain biopsy punches by Gba1 dPCR and normalized to the Tubb3 gene copy number to obtain VG copies per cell (B). Transgene (mRNA) expression measured by GBA1 RT-dPCR normalized to endogenous Hprt gene (C). Median with inter-quartile range across 64 punches representing 19 grey matter and 7 white matter regions. (D) Correlation of vector genome with transgene expression (mRNA) was determined between WT-GBA1 and SS3-GBA1. Each data point is average of all NHPs for that punch. Non- parametric Spearman’s rank correlation. (E) Lyso-GL1 changes in plasma pre-AAV, post-AAV and pre-CBE, and at necropsy across all NHPs. (F and G) Lyso-GL1 level (F) and C18 GL1 level (G) across 64 brain biopsy punches. Each data point is average of all NHPs in the group for that punch. ***p<0.001; Two way-ANOVA with Tukey’s multiple comparisons test. (H and I) Multiplex fluorescent imaging assay with in situ hybridization for mRNA and immunohistochemistry for GBA1 protein and cell marker. Low magnification image showing stained motor cortex region of SS3-GBA1 injected NHP and zoomed-in images corresponding to the white box (H). Triplex to determine identity of cross-corrected cells using specific cell type markers, NeuN for neurons, S100b for astrocytes, and Iba1 for microglia (I). Yellow arrowheads point to AAV-transduced cells and red arrowheads point to cross-corrected cells. Scale bar is 20 microns.

Article Snippet: The slides were then incubated with human GBA1 antibodies (1:100, abcam, ab125065 and 1:400, Novus, NBP2-45829) for rodent and NHP, respectively.

Techniques: Plasmid Preparation, Injection, IV Injection, Expressing, Clinical Proteomics, Multiplex Assay, Imaging, In Situ Hybridization, Immunohistochemistry, Marker, Staining

Journal: bioRxiv

Article Title: AAV gene therapy for GBA-PD and Gaucher Disease

doi: 10.1101/2025.06.17.660133

Figure Lengend Snippet: (A) Study design: 2-3 years old cynomolgus monkeys (2-3kg) were administered with 3 different doses of AAV GMU01-SS3-GBA1 by direct injection to the cisterna magna (iCM) in 2.5 ml volume at 0.125 ml/min rate, 2.5e12 vector genomes per animal (low dose), 7.5e12 vector genomes per animal (mid dose), and 2.5e13 vector genomes per animal (high dose). 8 weeks post-dosing, 30 mg/kg of CBE was administered by IV injection 24 hours prior to necropsy. Samples represent 64 brain biopsy punches encompassing 19 distinct grey matter regions and 7 distinct white matter regions. (B and C) Assessment of vector genomes in dose-range finding study with 3 doses tested, 5 NHPs per group. Median with inter-quartile range across 64 punches representing 19 grey matter and 7 white matter regions (B). Same data shown across different brain regions (C). T/I/C: temporal/insulate/cingulate (D) GCase enzyme activity in dose-range finding study with 3 doses tested, N=5 NHPs per group. Median with inter-quartile range across 64 punches representing 19 grey matter and 7 white matter regions. **p<0.05, ***p<0.001; Two way-ANOVA with Tukey’s multiple comparisons test. (E and F) Lipidomics performed in 3 NHPs per group. Lyso-GL1 level measured in plasma (E) and from 47 brain biopsy punches from 19 grey matter regions. Median with inter-quartile range across punches. ****p<0.0001, ***p<0.001; Two way-ANOVA with Tukey’s multiple comparisons test. (G – J) Histopathological analyses in NHPs with AAV.GMU01 SS3-GBA1 dose-range finding study. Histopathological findings reported with severity scores across both central nervous system and peripheral tissues by board certified clinician. Scores reported for brain (G), spinal cord (H), DRGs (I), and sciatic nerve (J). Data are Mean ± SEM. Each dot is score for individual NHP. (K) Quantification of human GBA1 protein levels in 12 brain regions from N=10 healthy human donor brain tissues (aged 55-to-75 years old) by LC-MS. Human tissue was obtained from the NIH Neurobiobank at the University of Maryland, Baltimore, MD and the Sepulveda Research Corporation. Data are Mean ± SEM. Each data point represents brain biopsy punches from each human donor. CC: corpus callosum; SN: substantia nigra; DN: dentate nucleus; Th: Thalamus, lateral nuclear group; Cb: cerebellum; PWM: periventricular white matter; Hp: hippocampus; BA: Brodmann area. (L) Quantification of human GBA1 protein levels from 3 NHPs per group. Median with inter- quartile range across 32 punches from NHPs plotted against the 12 punches from human donors. ***p<0.0001; Two way-ANOVA with Tukey’s multiple comparisons test.

Article Snippet: The slides were then incubated with human GBA1 antibodies (1:100, abcam, ab125065 and 1:400, Novus, NBP2-45829) for rodent and NHP, respectively.

Techniques: Injection, Plasmid Preparation, IV Injection, Activity Assay, Clinical Proteomics, Liquid Chromatography with Mass Spectroscopy

Journal: bioRxiv

Article Title: AAV gene therapy for GBA-PD and Gaucher Disease

doi: 10.1101/2025.06.17.660133

Figure Lengend Snippet: (A) Study design: AAV GMU01-SS3-GBA1 was administered by bilateral ICV to 4-month-old Gba D409V/+ mice at 1.6e11 vector genomes per animal and 4μl per hemisphere. 6-12 animals per group. Brain, plasma, and CSF were analyzed 3-month, 6-month, and 9-month post-injection. CBE (conduritol β-epoxide) was administered at 100 mg/kg 24 hours prior to each timed necropsy. (B) Representative image of in situ hybridization to WPRE mRNA from the analyzed sagittal sections. (C – F) Vector genome assessment of the longitudinal pharmacology study with in-life duration of 3-month, 6-month, and 9-month post AAV-dosing. AAV vector genome copies (left) and Lyso- GL1 lipid clearance (right) in cortex (C), sub-cortex (D), cerebellum (E), and hindbrain (F) across all mice in the study. 6 animals for No CBE group, 12 animals for Vehicle group, and 12 animals for SS3-GBA1 group. Data are Mean ± SEM. ***p<0.0001; Two way-ANOVA with Tukey’s multiple comparisons test. (G) Lyso-GL1 lipid clearance in plasma of SS3-GBA1 injected mice. Data is Mean ± SEM. ***P<0.0001; Two way-ANOVA with Tukey’s multiple comparisons test.

Article Snippet: The slides were then incubated with human GBA1 antibodies (1:100, abcam, ab125065 and 1:400, Novus, NBP2-45829) for rodent and NHP, respectively.

Techniques: Plasmid Preparation, Clinical Proteomics, Injection, In Situ Hybridization

Journal: bioRxiv

Article Title: AAV gene therapy for GBA-PD and Gaucher Disease

doi: 10.1101/2025.06.17.660133

Figure Lengend Snippet: (A) Study design: 3-month-old C57/BL6 mice were injected with 4e13 VG/kg of vehicle or AAV GMU01-SS3-GBA1 intravenously. AAVs were expressed for 4 weeks followed by 100mg/kg of CBE IP injection 24 hours prior to necropsy. (B) Vector genome assessed in different visceral organs such as liver, spleen, heart, and soleus muscle. 8 animals per group. Data is Mean ± SEM. (C) Schematic view of liver sectioning strategy and representative images demonstrating robust GBA1 secretion. Vector biodistribution is shown with in situ hybridization to WPRE (top panels) and GBA1 protein expression is shown with huGBA1 immunohistochemistry after 4 weeks of expression. (D – G) Quantification of human GBA1 protein level by ELISA and Lyso-GL1 lipid clearance by LC-MS in liver (D), spleen (E), heart (F), and soleus muscle (G). 8 animals per group, No CBE group in the graphs shown is control mice that did not receive any AAV vector or CBE injection. Data are Mean ± SEM. *p<0.05, ***p<0.0001; unpaired Student’s t-test (human GBA1 protein) and ***p<0.0001; One way-ANOVA with Dunnett’s multiple comparisons test (Lyso-GL1). All groups compared to vehicle group. (H) Evaluation of vector exposure, GCase enzyme activity, and Lyso-GL1 lipid clearance from bone marrow. 8 animals per group and No CBE group in the graphs shown is control mice that did not receive any vector or CBE injection. Data are Mean ± SEM. ***p<0.0001; unpaired Student’s t-test (VGs/cell) and ***p<0.001; One way-ANOVA with Tukey’s multiple comparisons test (GCase activity and Lyso-GL1).

Article Snippet: The slides were then incubated with human GBA1 antibodies (1:100, abcam, ab125065 and 1:400, Novus, NBP2-45829) for rodent and NHP, respectively.

Techniques: Injection, Plasmid Preparation, In Situ Hybridization, Expressing, Immunohistochemistry, Enzyme-linked Immunosorbent Assay, Liquid Chromatography with Mass Spectroscopy, Control, Activity Assay

Journal: Nature Communications

Article Title: Exosomal transfer of stroma-derived miR21 confers paclitaxel resistance in ovarian cancer cells through targeting APAF1

doi: 10.1038/ncomms11150

Figure Lengend Snippet: ( a ) Transcriptome profiling of SKOV3 cells transfected with miR21 precursor or control miR. A heatmap shows the top 10 miR21 regulated genes related to chemoresistance and metastasis. ( b ) Pathway analysis showing chemoresistance-related genes that were upregulated (red) or downregulated (green) in miR21-transfected SKOV3 cells. ( c ) SKOV3 cells incubated with CAA- and CAF-derived exosomes showed lower APAF1 expression than did controls. The results were the average from at least three independent experiments. Mean±s.d.; *** P <0.001; two-tailed Student's t -test. ( d ) Immunolocalization of APAF1 on ovarian tumour tissue sections ( n =5) demonstrated lower APAF1 level in the stromal–epithelial interface compared with that in the centre of the tumour tissues. A representative serial section of the tumour tissue was stained with haematoxylin and eosin (right panel). Dotted line and arrowheads indicate the stromal–epithelial interface between CAF and tumour (T). Scale bar, 10 μm. ( e , f ) Ovarian cancer cells transfected with miR21 precursor had lower APAF1 mRNA and protein levels than did controls. The results were the average from at least three independent experiments. Mean±s.d.; *** P <0.001 and ** P <0.01; two-tailed Student's t -test. ( g ) The correlation between miR21 and APAF1 mRNA levels in microdissected ovarian cancer tissues was determined using a Spearman's correlation analysis. ( h ) Recurrent ovarian cancer PEA2 cells expressed lower APAF1 expression than did primary PEA1 cells. The results were the average from at least three independent experiments. Mean±s.d.; ** P <0.01; two-tailed Student's t -test. ( i ) A consensus miR21-binding site was identified within the coding sequence of APAF1. ( j ) Co-transfection of pre-miR21 and luciferase vector with APAF1 coding sequence decreased the luciferase expression in SKOV3 cells in a dose-dependent manner. The results were the average from at least three independent experiments. Mean±s.d.; *** P <0.001, ** P <0.01 and * P <0.05; two-tailed Student's t -test. ( k ) The luciferase activity was measured following co-transfection with either wild-type (WT) or mutated (Mut) APAF1 coding sequence vectors (mutated sequence shown in red in upper panel). The results were the average from at least three independent experiments. Mean±s.d.; *** P <0.001 and ** P <0.01; two-tailed Student's t -test.

Article Snippet: The expression of APAF1 was determined by multiplexing quantitative PCR (TaqMan Gene Expression Assay) using FAM-labelled APAF1 (Hs00559441_m1) together with VIC-labelled glyceraldehyde 3-phosphate dehydrogenase-specific TaqMan probes and primers.

Techniques: Transfection, Control, Incubation, Derivative Assay, Expressing, Two Tailed Test, Staining, Binding Assay, Sequencing, Cotransfection, Luciferase, Plasmid Preparation, Activity Assay

Journal: Nature Communications

Article Title: Exosomal transfer of stroma-derived miR21 confers paclitaxel resistance in ovarian cancer cells through targeting APAF1

doi: 10.1038/ncomms11150

Figure Lengend Snippet: ( a ) Overexpression of APAF1 by full-length transfection increased paclitaxel sensitivity in ovarian cancer SKOV3 and OVCA432 cells. The results were the average from at least three independent experiments. Mean±s.d.; ** P <0.01 and * P <0.05; two-tailed Student's t -test. ( b ) Co-transfection of miR21 precursor and full-length APAF1 decreased paclitaxel resistance compared with the co-transfection of pre-miR21 and the control vector in both SKOV3 and OVCA432 cells. The results were the average from at least three independent experiments. Mean±s.d.; ** P <0.01 and * P <0.05; two-tailed Student's t -test. ( c – e ) APAF1 overexpression enhanced the paclitaxel sensitivity of ovarian cancer cells in vivo . APAF1 stably overexpressing ovarian cancer OVCA432 cells were generated using the lentiviral transduction method and were intraperitoneally injected into female BALB/c athymic nude mice, followed by paclitaxel treatment. The tumour volumes were measured and quantified using the IVIS-Lumina XR in vivo imaging system after a 2-week 5 mg kg −1 paclitaxel treatment. ( c ) Box plot showing a significant decrease in luciferase activity in the APAF1 overexpression group ( n =7) compared with the control group ( n =7) after paclitaxel treatment ( P =0.038; Mann–Whitney U -test). ( d ) Representative images show a decrease in luminescence in the APAF1 overexpression group compared with the control group after paclitaxel treatment. ( e ) Immunolocalization of APAF1 on paraffinized sections of tumour tissues collected from mice demonstrated a higher APAF1 level in the APAF1 overexpression group ( n =7) compared with the control group ( n =7). Representative microscopic images were illustrated. Scale bar, 10 μm.

Article Snippet: The expression of APAF1 was determined by multiplexing quantitative PCR (TaqMan Gene Expression Assay) using FAM-labelled APAF1 (Hs00559441_m1) together with VIC-labelled glyceraldehyde 3-phosphate dehydrogenase-specific TaqMan probes and primers.

Techniques: Over Expression, Transfection, Two Tailed Test, Cotransfection, Control, Plasmid Preparation, In Vivo, Stable Transfection, Generated, Transduction, Injection, In Vivo Imaging, Luciferase, Activity Assay, MANN-WHITNEY