Journal: Biomedicines

Article Title: SOX9 Protein in Pancreatic Cancer Regulates Multiple Cellular Networks in a Cell-Specific Manner

doi: 10.3390/biomedicines10071466

Figure Lengend Snippet: SOX9 protein and SOX9 mRNA expression in AsPC-1, BxPC-3, Colo357, Capan-2, MiaPaCa-2, and Panc1 cells. ( A ) Western blot analysis of the expression of E-cadherin (CDH1), cytokeratin-19 (KRT19), vimentin (VIM), SOX9, and GAPDH in the investigated cell lines. Pancreatic cancer cells were seeded in 6-well plates (0.5 × 10 6 cells per well). After 48 h, cell lysates were prepared by adding SDS sample buffer (200 µL per well). ( B ) Densitometric quantitation of Western blots from ( A ). The levels of the SOX9 protein were normalized to the levels of the GAPDH protein. ( C ) Real-time qPCR analysis of the mRNA expression of SOX9 in pancreatic cancer lines. The results are presented as the mean ± SEM of three independent experiments and normalized against the HPRT expression. ( D ) Immunofluorescence imaging of the SOX9 expression in Panc1 cells. Cells were stained for total cytokeratin (green) and SOX9 (red). Nuclei were stained with DAPI (blue). Scale bar = 100 µm.

Article Snippet: AsPC-1, BxPC-3, Capan-2, Mia PaCa-2, and Panc1 human pancreatic cancer cell lines were obtained from the American Type Cell Collection (ATCC, Manassas, VA, USA), and Colo357 [ ] was obtained from Dr. Klaus Felix (University Hospital Heidelberg, Heidelberg, Germany).

Techniques: Expressing, Western Blot, Quantitation Assay, Immunofluorescence, Imaging, Staining

Journal: Biomedicines

Article Title: SOX9 Protein in Pancreatic Cancer Regulates Multiple Cellular Networks in a Cell-Specific Manner

doi: 10.3390/biomedicines10071466

Figure Lengend Snippet: The effect of SOX9 downregulation on the expression levels of protein markers of differentiation and developmental transcription factors in pancreatic cancer cells. ( A ) Western blot analysis of the expression of SOX9 in AsPC-1, BxPC-3, Colo357, Capan-2, MiaPaCa-2, and Panc1 cells transfected with control siNeg and siSOX9 ( n = 3). GAPDH and beta-tubulin (TUBB) were used as loading and normalization controls. ( B ) Immunofluorescence imaging of the SOX9 expression in Panc1 cells transfected with control siNeg and siSOX9. Cells were stained for total cytokeratin (green) and SOX9 (red). Nuclei were stained with DAPI (blue). Scale bar = 50 µm. ( C ) Western blot analysis of the expression of epithelial cell protein markers in the investigated cell lines transfected with control siNeg and siSOX9 ( n = 3). ( D ) Heatmap of differentially expressed epithelial cell proteins from ( C ). Color code: red, upregulation; blue, downregulation; gray, no change. ND = undetected expression. ( E ) Western blot analysis of the expression of mesenchymal cell protein markers in the investigated cell lines transfected with control siNeg and siSOX9 ( n = 3). ( F ) Heatmap of differentially expressed mesenchymal cell proteins from ( E ). Color code: red, upregulation; blue, downregulation; gray, no change. ND = undetected expression. ( G ) Western blot analysis of the expression of developmental transcription factors in the investigated cell lines transfected with control siNeg and siSOX9 ( n = 3). ( H ) Heatmap of differentially expressed developmental transcription factors from ( G ). Color code: red, upregulation; blue, downregulation; gray, no change. ND = undetected expression.

Article Snippet: AsPC-1, BxPC-3, Capan-2, Mia PaCa-2, and Panc1 human pancreatic cancer cell lines were obtained from the American Type Cell Collection (ATCC, Manassas, VA, USA), and Colo357 [ ] was obtained from Dr. Klaus Felix (University Hospital Heidelberg, Heidelberg, Germany).

Techniques: Expressing, Western Blot, Transfection, Control, Immunofluorescence, Imaging, Staining

Journal: Biomedicines

Article Title: SOX9 Protein in Pancreatic Cancer Regulates Multiple Cellular Networks in a Cell-Specific Manner

doi: 10.3390/biomedicines10071466

Figure Lengend Snippet: The effect of SOX9 downregulation on the expression levels of cell cycle protein regulators. ( A ) Western blot analysis of the expression of cell cycle protein regulators in the investigated cell lines transfected with control siNeg and siSOX9 ( n = 3). ( B ) Heatmap of differentially expressed cell cycle protein regulators from ( B ). Color code: red, upregulation; blue, downregulation; gray, no change. ND = undetected expression. ( C ) Immunofluorescence imaging of p21 Waf1/Cip1 (CDKN1A) expression in Colo357 and Panc1 cells transfected with control siNeg and siSOX9. Cells were stained for SOX9 (red). Nuclei were stained with DAPI (blue). Scale bar = 50 μm. ( D , E ) Relative Western blot, RT-qPCR, and RNA-Seq data quantification of the levels of SOX9, SNAI1, SNAI2, FOXA2, GATA4, CDKN1A, TP53, and PTEN in control siNeg and siSOX9 transfected Colo357 ( D ) and Panc1 ( E ) cells. RT-qPCR and RNA-Seq data are means ± SEM from three technical replicates and representative of at least three experiments. * p ≤ 0.05; ** p ≤ 0.01 compared with siNeg control.

Article Snippet: AsPC-1, BxPC-3, Capan-2, Mia PaCa-2, and Panc1 human pancreatic cancer cell lines were obtained from the American Type Cell Collection (ATCC, Manassas, VA, USA), and Colo357 [ ] was obtained from Dr. Klaus Felix (University Hospital Heidelberg, Heidelberg, Germany).

Techniques: Expressing, Western Blot, Transfection, Control, Immunofluorescence, Imaging, Staining, Quantitative RT-PCR, RNA Sequencing

Journal: Biomedicines

Article Title: SOX9 Protein in Pancreatic Cancer Regulates Multiple Cellular Networks in a Cell-Specific Manner

doi: 10.3390/biomedicines10071466

Figure Lengend Snippet: Effect of SOX9 expression on cell proliferation, cellular senescence, apoptosis, and cell migration. ( A ) Effect of SOX9 downregulation on the cell proliferation of BxPC-3, Colo357, MiaPaCa-2, and Panc1. Pancreatic cancer cells seeded in 6-well plates were transfected with siSOX9 and control siNeg. After 72 h, the cell number was measured by counting cells using the TC20 automated cell counter. Data were expressed as the percentage from control cell growth. Data are means ± SEM from three technical replicates and representative of at least three experiments. * p ≤ 0.05; ** p ≤ 0.01 compared with siNeg control. ( B ) Effect of SOX9 downregulation on the senescence-associated β-galactosidase (SA-β-Gal) activity of BxPC-3, Colo357, MiaPaCa-2, and Panc1 cells. SA-β-Gal activity data were expressed relative to SA-β-Gal activity levels in siNeg and siSOX9 transfected cells. ( C ) Effect of SOX9 downregulation on caspase 3/7, caspase 8, and caspase 9 activities of BxPC-3, Colo357, MiaPaCa-2, and Panc1 cells. Caspase activity data were expressed relative to caspase activity levels in siNeg and siSOX9 transfected cells, respectively. ( D ) Panc1 cells were transfected with siSOX9 and siNeg for 72 h and then analyzed by annexin V–AF488/DAPI staining with flow cytometry analysis. The lower right area (Q3) shows early apoptotic cells, and the upper right area (Q2) shows late apoptotic cells. ( E ) Summary graphs of the flow cytometry results. ( F ) Western blot analysis of SOX9 and CDKN1A expression in Panc1 cells transfected with pCMV6 empty plasmid and pCMV6-SOX9-FLAG after 72 h post-transfection. GAPDH was used as control. ( G ) Effect of SOX9 upregulation on cell proliferation of BxPC-3 and Colo357. Pancreatic cancer cells seeded in 6-well plates were transfected with empty pCMV6 vector and pCMV6-SOX9-FLAG. After 72 h, the cell number was measured by counting cells using the TC20 automated cell counter. ( H ) Western blot analysis of the expression of SOX9, TP53, CDKN1A, and CDKN1B in Panc1 cells transfected with control siNeg and siSOX9. Cell lysates were prepared at 72, 96, and 120 h after first siRNA transfection. GAPDH was used as loading control. ( I ) The representative images displayed the GFP distributions of transplanted Panc1-EGFP control siNeg transfected cells in Danio rerio embryo. The microinjection site Danio rerio embryo is indicated with a red circle. The bioimaging of injected embryos was performed 2 days after cell transplantation. PH, phase contrast; FL, fluorescence. ( J ) The representative image displayed the GFP distributions of transplanted Panc1-EGFP siSOX9 transfected cells in Danio rerio embryo. The microinjection site Danio rerio embryo is indicated with a red circle. The bioimaging of injected embryos was performed 2 days after cell transplantation. PH, phase contrast; FL, fluorescence.

Article Snippet: AsPC-1, BxPC-3, Capan-2, Mia PaCa-2, and Panc1 human pancreatic cancer cell lines were obtained from the American Type Cell Collection (ATCC, Manassas, VA, USA), and Colo357 [ ] was obtained from Dr. Klaus Felix (University Hospital Heidelberg, Heidelberg, Germany).

Techniques: Expressing, Migration, Transfection, Control, Activity Assay, Staining, Flow Cytometry, Western Blot, Plasmid Preparation, Microinjection, Injection, Transplantation Assay, Fluorescence

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: Journal of nanobiotechnology

Article Title: Adipose-derived exosomes ameliorate skeletal muscle atrophy via miR-146a-5p/IGF-1R signaling.

doi: 10.1186/s12951-024-02983-7

Figure Lengend Snippet: Fig. 1 Effects of adipose-specific miR-146a-5p knockout on endurance exercise capacity, metabolism, and glucose homeostasis in mice. (a) The sche matic diagram for the development strategy of miR-146a-5p-knockout mice. (b) aKO mouse WAT tissue gDNA PCR result with a sequence of only 200 bp. (c) The expression of miR-146a-5p gene in BAT, iWAT, and eWAT of Flox and aKO mice (n = 6). (d) Body weight (n = 8). (e) Body composition (n = 8). (f) Representative images of body imaging. (g) Representative images of mice. (h) Representative H&E staining of iWAT, eWAT, and BAT from mice (scale bar = 50 μm). (i) Tissue weight in BAT, iWAT, eWAT, GAS, SOL, TA, and EDL of mice (n = 7). (j) Running distance at low speed (n = 6). (k) Score of weight lifting (n = 7). (l) Muscle grip strength (n = 7). (m) Representative cross sections TA fiber immunofluorescent MyHC staining (scale bar = 100 μm). (n) Frequency histogram of fiber cross-sectional area (n = 6). (o) RT-qPCR analysis for Cyclin A2, Cyclin D1, Cyclin E1, PCNA, MyoD, MyoG, Fbx32, and MuFR in the TA muscles of Flox and aKO mice (n = 6). (p, q) The protein levels and statistical analyses of Cyclin A2, Cyclin D1, Cyclin E1, PCNA, MyHC, MyoD, MyoG, Fbx32, and MuFR measured by Western blot in the TA muscles of Flox and aKO mice (n = 3). (r, s) ELISA analysis for IL-6 and TNF-α in Flox and aKO mice (n = 6). (t, u) The O2 consumption (VO2) (n = 6). (v, w) RER (n = 6). (x, y) IPITT and IPGTT blood glucose changes in Flox and aKO mice (n = 8). Values are presented as means ± SEM, *P <0.05, and **P <0.01, according to the non-paired Student’s t-test or one-way ANOVA between individual groups

Article Snippet: Following a 1-hour blocking step using goat serum containing 5%, the cells were then incubated overnight at 4 °C with anti-MyHC (MAB4470, R&D System), and monoclonal anti-MyoD antibody (sc-377460, Santa Cruz).

Techniques: Knock-Out, Sequencing, Expressing, Imaging, Staining, Quantitative RT-PCR, Muscles, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Journal of nanobiotechnology

Article Title: Adipose-derived exosomes ameliorate skeletal muscle atrophy via miR-146a-5p/IGF-1R signaling.

doi: 10.1186/s12951-024-02983-7

Figure Lengend Snippet: Fig. 2 3T3-L1 cell-derived miR-146a-5p participates in C2C12 cell proliferation and differentiation. (a) 3T3-L1 cells were co-cultured with C2C12 cells, and the cells were grown in a transwell. (b) The expression of miR-146a-5p gene in 3T3-L1 cells following transfection with mimics and inhibitors (n = 6). (c) The expression of miR-146a-5p gene in co-cultured C2C12 cells following transfecting 3T3-L1 cells with mimics and inhibitors (n = 6). (d) CCK-8 result of co- cultured C2C12 cells (n = 9). (e, f) EdU image and statistical analyses of C2C12 cells (scale bar = 50 μm) (n = 7). (g) RT-qPCR analysis for Cyclin A2, Cyclin D1, Cyclin E1, and PCNA of C2C12 cells (n = 6). (h, i) The protein levels of Cyclin A2, Cyclin D1, Cyclin E1, and PCNA by Western blot and the statistical analyses results of C2C12 cells (n = 3). (j) RT-qPCR analysis fo MyoD, MyoG, Fbx32 and MuFR of C2C12 cells (n = 6). (k, l) The protein levels of MyHC, MyoD, MyoG, Fbx32, and MuFR by Western blot and the statistical analyses results of C2C12 cells (n = 3). (m, n) Representative muscle fiber immunofluorescent MyHC staining of C2C12 cells (scale bar = 50 μm) (n = 4). Values are presented as means ± SEM, *P <0.05, and **P <0.01, according to the non-paired Student’s t-test or one-way ANOVA between individual groups

Article Snippet: Following a 1-hour blocking step using goat serum containing 5%, the cells were then incubated overnight at 4 °C with anti-MyHC (MAB4470, R&D System), and monoclonal anti-MyoD antibody (sc-377460, Santa Cruz).

Techniques: Derivative Assay, Cell Culture, Expressing, Transfection, CCK-8 Assay, Quantitative RT-PCR, Western Blot, Staining

Journal: Journal of nanobiotechnology

Article Title: Adipose-derived exosomes ameliorate skeletal muscle atrophy via miR-146a-5p/IGF-1R signaling.

doi: 10.1186/s12951-024-02983-7

Figure Lengend Snippet: Fig. 3 miR-146a-5p inhibits proliferation and promotes differentiation in C2C12 cells. (a) RT-qPCR to detect the expression of miR-146a-5p in proliferating C2C12 cells (n = 12). (b) The expression of miR-146a-5p gene in proliferating C2C12 cells after transfection with mimics and inhibitors (n = 12). (c, d) EdU image and statistical analyses of C2C12 (scale bar = 50 μm) (n = 12). (e) CCK-8 result of C2C12 (n = 8). (f, g) Cell cycle analysis of C2C12 by flow cytometry and statistical results (n = 3). (h) RT-qPCR analysis for Cyclin A2, Cyclin B1, Cyclin D1, PCNA and P21 in C2C12 (n = 6). (i, j) The protein levels of Cyclin A2, Cyclin D1, Cyclin E1, and PCNA by Western blot and the statistical analyses results in C2C12 (n = 3). (k) The expression of miR-146a-5p gene in differentiat ing C2C12 (n = 12). (l-n) Representative muscle fiber immunofluorescent MyHC staining in C2C12 (scale bar = 100 μm) (n = 4). (o) RT-qPCR analysis for MyoD, MyoG, Pax7, Fbx32, and MuFR in C2C12 (n = 9). (p-q) The protein levels of MyHC, MyoD, MyoG, Fbx32, and MuFR by Western blot and the statistical analyses results in C2C12 (n = 3). (r) RT-qPCR analysis for MyHC I, MyHC IIa, MyHC IIb and MyHC IIx in C2C12 (n = 9). (s) RT-qPCR analysis for IL-1β, IL-6 and TNF-α in C2C12 (n = 9). Values are presented as means ± SEM, *P <0.05, and **P <0.01, according to the non-paired Student’s t-test or one-way ANOVA between individual groups

Article Snippet: Following a 1-hour blocking step using goat serum containing 5%, the cells were then incubated overnight at 4 °C with anti-MyHC (MAB4470, R&D System), and monoclonal anti-MyoD antibody (sc-377460, Santa Cruz).

Techniques: Quantitative RT-PCR, Expressing, Transfection, CCK-8 Assay, Cell Cycle Assay, Flow Cytometry, Western Blot, Staining

Journal: Journal of nanobiotechnology

Article Title: Adipose-derived exosomes ameliorate skeletal muscle atrophy via miR-146a-5p/IGF-1R signaling.

doi: 10.1186/s12951-024-02983-7

Figure Lengend Snippet: Fig. 4 miR-146a-5p reversed the myotube atrophy of C2C12 cells induced by aKO-WAT-Exos. (a) Electron microscopy results and nanoparticle tracking analysis was used to determine the size distribution of adipose-derived exosomes (scale bar = 200 nm). (b) Calnexin in adipose cells and Alix, TSG101, CD9, and CD63 in adipose-derived exosomes of aKO and Flox mice were detected by Western Blot. (c) The expression of miR-146a-5p gene in Flox-iWAT- Exos, aKO-iWAT-Exos, Flox-eWAT-Exos, aKO-eWAT-Exos (n = 6). (d) CCK-8 result of C2C12 cells treated with Control (PBS), Flox-iWAT-Exos, aKO-iWAT-Exos, Flox-eWAT-Exos, aKO-eWAT-Exos (n = 8). (e, f) EdU and statistical analyses image of C2C12 (scale bar = 50 μm) (n = 6). (g) RT-qPCR analysis for Cyclin A2, Cyclin D1, Cyclin E1, PCNA in C2C12 cells (n = 6). (h) The protein levels of Cyclin A2, Cyclin D1, Cyclin E1, and PCNA by Western blot in C2C12 cells (n = 3). (i-k) Representative muscle fiber immunofluorescent MyHC staining, myotube diameter, and myotube fusion rate in C2C12 cells (scale bar = 100 μm) (n = 6). (l) RT-qPCR analysis for MyoD, MyoG, Fbx32, and MuFR in differentiated C2C12 (n = 6). (m) The protein levels of MyHC, MyoD, MyoG, Fbx32, and MuFR by Western blot in differentiated C2C12 (n = 3). (n) The expression of miR-146a-5p gene in proliferating C2C12 treated with aKO-WAT-Exos + NC and aKO-WAT-Exos + Mimics (n = 6). (o) CCK-8 result of C2C12 (n = 8). (p, q) EdU image and statistical results of C2C12 (scale bar = 50 μm) (n = 6). (r) RT-qPCR analysis for Cyclin A2, Cyclin D1, Cyclin E1, PCNA in C2C12 cells (n = 6). (s) The protein levels of Cyclin A2, Cyclin D1, Cyclin E1, PCNA in C2C12 (n = 3). (t, u) Representative muscle fiber immunofluorescent MyHC staining in C2C12 (n = 4) (scale bar = 50 μm). (v) RT-qPCR analysis for MyoD, MyoG, Fbx32 and MuFR in C2C12 (n = 6). (w) The protein levels of MyHC, MyoD, MyoG, Pax7, Fbx32, and MuFR by Western blot in C2C12 (n = 3). Values are presented as means ± SEM, *P <0.05, and **P <0.01, according to the non-paired Student’s t-test or one-way ANOVA between individual groups

Article Snippet: Following a 1-hour blocking step using goat serum containing 5%, the cells were then incubated overnight at 4 °C with anti-MyHC (MAB4470, R&D System), and monoclonal anti-MyoD antibody (sc-377460, Santa Cruz).

Techniques: Electron Microscopy, Derivative Assay, Western Blot, Expressing, CCK-8 Assay, Control, Quantitative RT-PCR, Staining

Journal: Journal of nanobiotechnology

Article Title: Adipose-derived exosomes ameliorate skeletal muscle atrophy via miR-146a-5p/IGF-1R signaling.

doi: 10.1186/s12951-024-02983-7

Figure Lengend Snippet: Fig. 5 WAT-derived exosomes miR-146a-5p may be involved in muscle atrophy. (a) The fluorescence signal distribution of PKH67-labeled adipose- derived exosomes in aKO organs for 24 h after TA injection. The isolated organs from left to right are as follows: heart, liver, spleen, lung, kidney, BAT, iWAT, eWAT, GAS, SOL, TA, EDL, and intestinal fat. (b) The expression of miR-146a-5p gene in aKO TA muscles 12 h after injected with aKO-Exos and Flox-Exos (n = 6). (c) RT-qPCR analysis for Cyclin A2, Cyclin D1, Cyclin E1, PCNA, MyoD, MyoG, Fbx32, and MuFR in aKO TA muscles (12 h) (n = 6). (d) The protein levels of Cyclin A2, Cyclin D1, Cyclin E1, PCNA, MyHC, MyoD, MyoG, Fbx32, and MuFR were measured by Western blot in aKO TA muscles (12 h) (n = 3). (e) The expression of miR-146a-5p gene in aKO TA muscles 24 h after injected with aKO-Exos and Flox-Exos (n = 6). (f) RT-qPCR analysis for Cyclin A2, Cyclin D1, Cyclin E1, PCNA, MyoD, MyoG, Fbx32, and MuFR in aKO mice TA muscles (24 h) (n = 6). (g) The protein levels of Cyclin A2, Cyclin D1, Cyclin E1, PCNA, MyHC, MyoD, MyoG, Fbx32, and MuFR were measured by Western blot in aKO TA muscles (24 h) (n = 3). (h) Body weight gain (n = 4). (i) Body composition (n = 4). (j) Representative images of body imaging. (k) Tissue weight in GAS, SOL, TA, and EDL of mice (n = 4). (l) Running distance at low speed (n = 3). (m) Score of weight lifting (n = 4). (n) Muscle grip strength (n = 4). (o) The expression of miR-146a-5p gene in the aKO TA muscles injected with aKO-Exos and Flox-Exos for 24 d (n = 6). (p) RT-qPCR analysis for Cyclin A2, Cyclin D1, Cyclin E1, PCNA, MyoD, MyoG, Fbx32, and MuFR in the aKO TA muscles injected with aKO-Exos and Flox-Exos for 24 d (n = 3). (q) The protein levels of Cyclin A2, Cyclin D1, Cyclin E1, PCNA, MyHC, MyoD, MyoG, Fbx32, and MuFR measured by Western blot in the aKO TA muscles injected with aKO-Exos and Flox-Exos for 24 d (n = 3). (r, s) Representative cross sections TA fiber immunofluorescent MyHC staining and statistical results in the aKO TA muscles injected with aKO-Exos and Flox-Exos for 24 d (scale bar = 100 μm) (n = 4). (t, u) Representative cross sections TA fiber immunofluorescent MyoD staining and statistical results in the aKO TA muscles injected with aKO-Exos and Flox-Exos for 24 d (scale bar = 100 μm) (n = 4). (v, w) Representative cross sections TA fiber immunofluorescent Pax7 staining and statistical results in the aKO TA muscles injected with aKO-Exos and Flox-Exos for 24 d (scale bar = 100 μm) (n = 4). Values are presented as means ± SEM, *P <0.05, and **P <0.01, according to the non-paired Student’s t-test or one-way ANOVA between individual groups

Article Snippet: Following a 1-hour blocking step using goat serum containing 5%, the cells were then incubated overnight at 4 °C with anti-MyHC (MAB4470, R&D System), and monoclonal anti-MyoD antibody (sc-377460, Santa Cruz).

Techniques: Derivative Assay, Fluorescence, Labeling, Injection, Isolation, Expressing, Muscles, Quantitative RT-PCR, Western Blot, Imaging, Staining

Journal: Journal of nanobiotechnology

Article Title: Adipose-derived exosomes ameliorate skeletal muscle atrophy via miR-146a-5p/IGF-1R signaling.

doi: 10.1186/s12951-024-02983-7

Figure Lengend Snippet: Fig. 6 miR-146a-5p alleviates muscle atrophy in vitro. (a, b) Representative muscle fiber immunofluorescent MyHC staining of CTX-induced C2C12 cells after transfection with miR-146a-5p mimics/inhibitors or co-treatment with Flox-Exos, aKO-Exos (scale bar = 100 μm) (n = 4). (c, d) Representative muscle fiber immunofluorescent MyoD staining of CTX-induced C2C12 cells after transfection with miR-146a-5p mimics/inhibitors or co-treatment with Flox- Exos, aKO-Exos (scale bar = 100 μm) (n = 4). (e, f) Representative muscle fiber immunofluorescent Pax7 staining of CTX-induced C2C12 cells after transfec tion with miR-146a-5p mimics/inhibitors or co-treatment with Flox-Exos, aKO-Exos (scale bar = 100 μm) (n = 4). Values are presented as means ± SEM, *P <0.05, and **P <0.01, according to the non-paired Student’s t-test or one-way ANOVA between individual groups

Article Snippet: Following a 1-hour blocking step using goat serum containing 5%, the cells were then incubated overnight at 4 °C with anti-MyHC (MAB4470, R&D System), and monoclonal anti-MyoD antibody (sc-377460, Santa Cruz).

Techniques: In Vitro, Staining, Transfection

Journal: Journal of nanobiotechnology

Article Title: Adipose-derived exosomes ameliorate skeletal muscle atrophy via miR-146a-5p/IGF-1R signaling.

doi: 10.1186/s12951-024-02983-7

Figure Lengend Snippet: Fig. 7 Adipose-derived miR-146a-5p is indispensable in muscle atrophy and repair. (a) RT-qPCR of miR-146a-5p in TA muscle cross-sections from injured (5, 7, and 15 days after CTX injection) Flox and aKO mice (n = 4). (b, c) Representative of MyHC immunofluorescent staining of TA muscle cross-sections from uninjured (Day 0) and injured (5, 7, and 15 days after CTX injection) Flox and aKO mice (scale bar = 100 μm) (n = 4). (d, e) Representative of MyoD immunofluorescent staining of TA muscle cross-sections from uninjured and injured Flox and aKO mice (scale bar = 100 μm) (n = 4). (f, g) Representative of Pax7 immunofluorescent staining of TA muscle cross-sections from both uninjured and injured Flox and aKO mice (scale bar = 100 μm) (n = 4). (h) RT-qPCR analysis for Cyclin A2, Cyclin D1, Cyclin E1, and PCNA in TA muscle cross-sections from injured Flox and aKO mice (n = 4). (i) RT-qPCR analysis for MyoD, MyoG, Pax7, Fbx32, and MuFR in TA muscle cross-sections from injured (5, 7, and 15 days after CTX, aKO-Exos and Flox-Exos injection) aKO mice (n = 4). (j) The protein levels of MyHC, MyoD, MyoG, Pax7, Fbx32, and MuFR measured by Western blot in TA muscle cross-sections from injured aKO mice (n = 3). (k) RT-qPCR of miR-146a-5p in TA muscle cross-sections from injured aKO mice treated with Exos (aKO-Exos and Flox-Exos injection) (n = 4). (l, m) Representative of MyHC immunofluorescent staining of TA muscle cross-sections from both uninjured and injured aKO mice treated with Exos (scale bar = 100 μm) (n = 4). (n, o) Representative of MyoD immunofluorescent staining of TA muscle cross-sections (scale bar = 100 μm) (n = 4). (p, q) Representative of Pax7 immunofluorescent staining of TA muscle cross-sections from both uninjured (Day 0) and injured (5, 7, and 15 days after CTX, aKO-Exos, and Flox- Exos injection) aKO mice (scale bar = 100 μm) (n = 4). (r) RT-qPCR analysis for Cyclin A2, Cyclin D1, Cyclin E1, and PCNA in TA muscle cross-sections from injured (5, 7, and 15 days after CTX, aKO-Exos, and Flox-Exos injection) aKO mice (n = 4). (s) RT-qPCR analysis for MyoD, MyoG, Pax7, Fbx32, and MuFR in TA muscle cross-sections from injured (5, 7, and 15 days after CTX, aKO-Exos and Flox-Exos injection) aKO mice (n = 4). (t) The protein levels of MyHC, MyoD, MyoG, Pax7, Fbx32, and MuFR measured by Western blot in TA muscle cross-sections from injured (5, 7, and 15 days after CTX, aKO-Exos, and Flox-Exos injection) aKO mice (n = 3). Values are presented as means ± SEM, *P <0.05, and **P <0.01, according to the non-paired Student’s t-test or one-way ANOVA between individual groups

Article Snippet: Following a 1-hour blocking step using goat serum containing 5%, the cells were then incubated overnight at 4 °C with anti-MyHC (MAB4470, R&D System), and monoclonal anti-MyoD antibody (sc-377460, Santa Cruz).

Techniques: Derivative Assay, Quantitative RT-PCR, Injection, Staining, Western Blot

Journal: Journal of nanobiotechnology

Article Title: Adipose-derived exosomes ameliorate skeletal muscle atrophy via miR-146a-5p/IGF-1R signaling.

doi: 10.1186/s12951-024-02983-7

Figure Lengend Snippet: Fig. 8 miR-146a-5p prevents muscle atrophy by targeting IGF-1R. (a) miR-146a-5p has a target interaction with the 3’UTR of IGF-1R. (b) Relative luciferase activity was calculated by firefly luminescence/renilla luminescence (WT: pmiGLO- IGF-1R -WT, mut site 1 + 2: pmirGLO-IGF-1R -Mut1 + pmirGLO-IGF-1R- Mut2, mut site 1: pmirGLO- IGF-1R -Mut1, mut site 2: pmirGLO-IGF-1R -Mut2) (n = 10). (c) The expression of miR-146a-5p gene in C2C12 cells following transfection with siRNA-IGF-1R and co-treatment with siRNA-IGF-1R and miR-146a-5p inhibitor (n = 4). (d) CCK-8 result of C2C12 cells (n = 8). (e, f) EdU image and statistical analyses of C2C12 cells (scale bar = 100 μm) (n = 6). (g) RT-qPCR analysis for Cyclin A2, Cyclin D1, Cyclin E1, and PCNA in C2C12 cells (n = 4). (h, i) The protein levels of Cyclin A2, Cyclin D1, Cyclin E1, and PCNA by Western blot and the statistical analyses results in C2C12 cells (n = 3). (j-l) Representative muscle fiber immunofluorescent MyHC staining, myotube diameter, and myotube fusion rate of C2C12 cells (scale bar = 100 μm) (n = 4). (m, n) Representative muscle fiber immunofluorescent MyoD staining and statistical results of C2C12 cells (scale bar = 100 μm) (n = 4). (o, p) Representa tive muscle fiber immunofluorescent Pax7 staining and statistical results of C2C12 cells (scale bar = 100 μm) (n = 4). (q) RT-qPCR analysis for IGF-1R, MyoD, MyoG, Pax7, Fbx32 and MuFR in C2C12 cells (n = 4). (r) The protein levels of MyHC, MyoD, MyoG, Pax7, Fbx32, and MuFR by Western blot in C2C12 cells. (s) The protein levels of IGF-1R, P-PI3K, PI3K, P-AKT, AKT, P-mTOR, mTOR, P-S6, S6, P-FoxO3 and FoxO3 measured by Western blot in C2C12 cells. (n = 3). (t) Immunoprecipitation assay revealed an enrichment of P-PI3K, PI3K, P-AKT, AKT, P-mTOR, mTOR, P-S6, S6, P-FoxO3 and FoxO3 when introduced with IGF- 1R. (u) The protein levels of IGF-1R, P-PI3K, PI3K, P-AKT, AKT, P-mTOR, mTOR, P-S6, S6, P-FoxO3, and FoxO3 measured by Western blot in the TA muscles of Flox and aKO mice (n = 3). (v) RT-qPCR analysis for IGF-1R in C2C12 transfected with miR-146a-5p mimics/inhibitor (n = 6). (w) The protein levels of IGF-1R, P-PI3K, PI3K, P-AKT, AKT, P-mTOR, mTOR, P-S6, S6, P-FoxO3 and FoxO3 by Western blot in C2C12 transfected with miR-146a-5p mimics/inhibitor (n = 3). (x) RT-qPCR analysis for IGF-1R in aKO TA muscles injected aKO-Exos and Flox-Exos for 24 d (n = 3). (y) The protein levels of IGF-1R, P-PI3K, PI3K, P-AKT, AKT, P-mTOR, mTOR, P-S6, S6, P-FoxO3, and FoxO3 measured by Western blot in aKO TA muscles injected aKO-Exos and Flox-Exos for 24 d (n = 3). (z) The protein levels of IGF-1R, P-PI3K, PI3K, P-AKT, AKT, P-mTOR, mTOR, P-S6, S6, P-FoxO3, and FoxO3 measured by Western blot in TA muscle cross-sections from injured (5, 7 and 15 days after CTX injection) Flox and aKO mice (n = 3). Values are presented as means ± SEM, *P <0.05, and **P <0.01, according to the non-paired Student’s t-test or one-way ANOVA between individual groups

Article Snippet: Following a 1-hour blocking step using goat serum containing 5%, the cells were then incubated overnight at 4 °C with anti-MyHC (MAB4470, R&D System), and monoclonal anti-MyoD antibody (sc-377460, Santa Cruz).

Techniques: Luciferase, Activity Assay, Expressing, Transfection, CCK-8 Assay, Quantitative RT-PCR, Western Blot, Staining, Immunoprecipitation, Muscles, Injection

Journal: International Journal of Nanomedicine

Article Title: Prostate-Specific Membrane Antigen and Esterase Dual Responsive Camptothecin–Oligopeptide Self-Assembled Nanoparticles for Efficient Anticancer Drug Delivery

doi: 10.2147/IJN.S331060

Figure Lengend Snippet: Schematic illustration of the PSMA and esterase dual responsive CPT nanoparticles (CPT-WT-H NPs) with negatively charged surface, prolonged blood circulation, excellent PSMA-targeted delivery, enhanced tumor accumulation, potent antitumor efficacy and low side effects.

Article Snippet: The in vitro PSMA response of the CPT-WT-H NPs was performed as described in the product catalog (Recombinant Human PSMA/FOLH1/NAALADase I, 4234-ZN, bio-techne, USA) with some modifications using LC-MS. CPT-WT-H NPs solutions (125 μL, 0.1 mg·mL −1 in HEPS buffer) were incubated with the rh-PSMA solution (125 μL, 1.0 μg·mL −1 in HEPS buffer) at 37°C for 2 h, 8 h, 12 h, 24 h and 48 h in a remonstrated oscillator (HZQ-QX, Harbin Donglian Instrument Co. Ltd., China).

Techniques:

Journal: International Journal of Nanomedicine

Article Title: Prostate-Specific Membrane Antigen and Esterase Dual Responsive Camptothecin–Oligopeptide Self-Assembled Nanoparticles for Efficient Anticancer Drug Delivery

doi: 10.2147/IJN.S331060

Figure Lengend Snippet: Characterization of CPT-WT-H NPs. ( A ) A TEM image of CPT-WT-H NPs dispersed in PBS (pH 7.4); inserts are the photographs of CPT-WT-H NPs in PBS in bright field (left) and under UV light at 365 nm with a red laser passing through (right). ( B ) DLS histograms of CPT-WT-H NPs in PBS. ( C ) Change of hydrodynamic diameter (Dh) of CPT-WT-H NPs in PBS for 120 h. ( D ) Schematic of sequential PSMA hydrolysis of CPT-WT-H (CPT12A-βAsp-GluγGluγGluγGlu) to yield the PSMA hydrolysate CPT12A-βAsp. ( E ) Characterization of in vitro PSMA response of CPT-WT-H NPs by LC-MS.

Article Snippet: The in vitro PSMA response of the CPT-WT-H NPs was performed as described in the product catalog (Recombinant Human PSMA/FOLH1/NAALADase I, 4234-ZN, bio-techne, USA) with some modifications using LC-MS. CPT-WT-H NPs solutions (125 μL, 0.1 mg·mL −1 in HEPS buffer) were incubated with the rh-PSMA solution (125 μL, 1.0 μg·mL −1 in HEPS buffer) at 37°C for 2 h, 8 h, 12 h, 24 h and 48 h in a remonstrated oscillator (HZQ-QX, Harbin Donglian Instrument Co. Ltd., China).

Techniques: In Vitro, Liquid Chromatography with Mass Spectroscopy

Journal: International Journal of Nanomedicine

Article Title: Prostate-Specific Membrane Antigen and Esterase Dual Responsive Camptothecin–Oligopeptide Self-Assembled Nanoparticles for Efficient Anticancer Drug Delivery

doi: 10.2147/IJN.S331060

Figure Lengend Snippet: In vitro cytotoxicity of CPT-WT-H NPs and CPT against PSMA-expressing (PSMA + ) cancer cell LNCaP-FGC ( A ) and non-PSMA-expressing (PSMA − ) cancer cells HepG2 ( B ), MCF-7 ( C ), HeLa ( D ), DU145 ( E ), PC-3 ( F ) and normal liver cells LO2 ( G ), the IC 50 values of CPT and CPT-WT-H NPs on different cell lines ( H ). *** p < 0.001, vs LNCaP-FGC cells.

Article Snippet: The in vitro PSMA response of the CPT-WT-H NPs was performed as described in the product catalog (Recombinant Human PSMA/FOLH1/NAALADase I, 4234-ZN, bio-techne, USA) with some modifications using LC-MS. CPT-WT-H NPs solutions (125 μL, 0.1 mg·mL −1 in HEPS buffer) were incubated with the rh-PSMA solution (125 μL, 1.0 μg·mL −1 in HEPS buffer) at 37°C for 2 h, 8 h, 12 h, 24 h and 48 h in a remonstrated oscillator (HZQ-QX, Harbin Donglian Instrument Co. Ltd., China).

Techniques: In Vitro, Expressing

Journal: International Journal of Nanomedicine

Article Title: Prostate-Specific Membrane Antigen and Esterase Dual Responsive Camptothecin–Oligopeptide Self-Assembled Nanoparticles for Efficient Anticancer Drug Delivery

doi: 10.2147/IJN.S331060

Figure Lengend Snippet: Cellular uptake of CPT-WT-H NPs in vitro. ( A ) CLSM images of LNCaP-FGC (PSMA + ) cells incubated with CPT-WT-H NPs (10 μM) for 1 and 4 h. ( B ) CLSM images of HepG2 (PSMA − ) cells incubated with CPT-WT-H NPs (10 μM) for 1 and 4 h. Nuclei were stained by PI (red), the blue color was indicative of CPT. ( C ) Mean fluorescence intensity of CPT-WT-H NPs internalized by LNCaP-FGC and HepG2 cells after incubation for 1, and 4 h. *** p < 0.001. ( D ) Flow cytometry analysis for apoptosis of LNCaP-FGC (PSMA + ) (1) and HepG2 (PSMA − ) (2) cells induced by CPT-WT-H NPs for 72 h. Q1, necrotic cells; Q2, late apoptotic cells; Q3, living cells; Q4, early apoptotic cells. Inserted numbers in the profiles indicate the percentage of the cells present in this area.

Article Snippet: The in vitro PSMA response of the CPT-WT-H NPs was performed as described in the product catalog (Recombinant Human PSMA/FOLH1/NAALADase I, 4234-ZN, bio-techne, USA) with some modifications using LC-MS. CPT-WT-H NPs solutions (125 μL, 0.1 mg·mL −1 in HEPS buffer) were incubated with the rh-PSMA solution (125 μL, 1.0 μg·mL −1 in HEPS buffer) at 37°C for 2 h, 8 h, 12 h, 24 h and 48 h in a remonstrated oscillator (HZQ-QX, Harbin Donglian Instrument Co. Ltd., China).

Techniques: In Vitro, Incubation, Staining, Fluorescence, Flow Cytometry

Journal: International Journal of Nanomedicine

Article Title: Prostate-Specific Membrane Antigen and Esterase Dual Responsive Camptothecin–Oligopeptide Self-Assembled Nanoparticles for Efficient Anticancer Drug Delivery

doi: 10.2147/IJN.S331060

Figure Lengend Snippet: In vivo NIRF imaging of DiR-doped CPT-WT-H NPs. ( A ) In vivo NIRF images of the MCF-7 tumor-bearing nude mice (PSMA-producing) after intravenous injection of free DiR (0.25 mg·mL −1 , 100 µL) or DiR-doped CPT-WT-H NPs (0.25 mg·mL −1 , 100 µL). ( B ) Integrated fluorescence intensity of tumor obtained after intravenous injection of DiR-doped CPT-WT-H NPs (0.25 mg·mL −1 , 100 µL). ( C ) Ex vivo NIRF images and ( D ) Integrated fluorescence intensity of the major organs and tumors excised at 48 h post intravenous injection of DiR-doped CPT-WT-H NPs, *** p < 0.001, vs tumor. ( E ) In vivo pharmacokinetics profiles of CPT-WT-H NPs s at the equivalent dose (30 mg·kg −1 ) in BALB/c mice. Error bars indicate SD (n = 6).

Article Snippet: The in vitro PSMA response of the CPT-WT-H NPs was performed as described in the product catalog (Recombinant Human PSMA/FOLH1/NAALADase I, 4234-ZN, bio-techne, USA) with some modifications using LC-MS. CPT-WT-H NPs solutions (125 μL, 0.1 mg·mL −1 in HEPS buffer) were incubated with the rh-PSMA solution (125 μL, 1.0 μg·mL −1 in HEPS buffer) at 37°C for 2 h, 8 h, 12 h, 24 h and 48 h in a remonstrated oscillator (HZQ-QX, Harbin Donglian Instrument Co. Ltd., China).

Techniques: In Vivo, Imaging, Injection, Fluorescence, Ex Vivo

Journal: International Journal of Nanomedicine

Article Title: Prostate-Specific Membrane Antigen and Esterase Dual Responsive Camptothecin–Oligopeptide Self-Assembled Nanoparticles for Efficient Anticancer Drug Delivery

doi: 10.2147/IJN.S331060

Figure Lengend Snippet: CPT-WT-H NPs significantly inhibited tumor progression and dramatically reduced side effects in an MCF-7 human breast cancer model. ( A ) The growth curves of MCF-7 tumor after treatment, which was initiated on day 13 post tumor inoculation, and drugs were intravenously injected every 2 days for 8 times (n = 6/group). ( B ) Changes of body weight in each group. ( C ) The tumor pictures in each group. ( D ) Excised tumor weight in each group. ( E ) Excised tumor volume in each group. * p < 0.05; *** p < 0.001. ( F ) Histological analysis of tumor sections stained with H&E.

Article Snippet: The in vitro PSMA response of the CPT-WT-H NPs was performed as described in the product catalog (Recombinant Human PSMA/FOLH1/NAALADase I, 4234-ZN, bio-techne, USA) with some modifications using LC-MS. CPT-WT-H NPs solutions (125 μL, 0.1 mg·mL −1 in HEPS buffer) were incubated with the rh-PSMA solution (125 μL, 1.0 μg·mL −1 in HEPS buffer) at 37°C for 2 h, 8 h, 12 h, 24 h and 48 h in a remonstrated oscillator (HZQ-QX, Harbin Donglian Instrument Co. Ltd., China).

Techniques: Injection, Staining

Journal: International Journal of Nanomedicine

Article Title: Prostate-Specific Membrane Antigen and Esterase Dual Responsive Camptothecin–Oligopeptide Self-Assembled Nanoparticles for Efficient Anticancer Drug Delivery

doi: 10.2147/IJN.S331060

Figure Lengend Snippet: Representative H&E-stained histological images obtained from liver ( A ) and kidneys ( B ) after intravenous administration of irinotecan (10 mg·kg −1 ) and CPT-WT-H NPs (10, 30, 60 mg·kg −1 ). The PBS-treated group and the untreated group were used as the control. (I: PBS, II: CPT-WT-H NPs (10mg·kg −1 ), III: CPT-WT-H NPs (30mg·kg −1 ), IV: CPT-WT-H NPs (60mg·kg −1 ), V: Irinotecan (10 mg·kg −1 ), VI: Normal group). ( C ) Blood biochemical analysis of after the antitumor efficacy evaluation. The PBS-treated group and the untreated group were used as the control. The main parameters of hepatic and renal function are alanine transaminase (ALT), aspartate transaminase (AST), blood urea nitrogen (BUN), and creatinine (CRE). Error bars indicate SD (n = 6). * p < 0.05; *** p < 0.001.

Article Snippet: The in vitro PSMA response of the CPT-WT-H NPs was performed as described in the product catalog (Recombinant Human PSMA/FOLH1/NAALADase I, 4234-ZN, bio-techne, USA) with some modifications using LC-MS. CPT-WT-H NPs solutions (125 μL, 0.1 mg·mL −1 in HEPS buffer) were incubated with the rh-PSMA solution (125 μL, 1.0 μg·mL −1 in HEPS buffer) at 37°C for 2 h, 8 h, 12 h, 24 h and 48 h in a remonstrated oscillator (HZQ-QX, Harbin Donglian Instrument Co. Ltd., China).

Techniques: Staining

Journal: Journal of Nanobiotechnology

Article Title: Lymph node-targeted neoantigen nanovaccines potentiate anti-tumor immune responses of post-surgical melanoma

doi: 10.1186/s12951-022-01397-7

Figure Lengend Snippet: Characterization and in vitro DC-activation of neoantigen nanovaccines. a 1 H NMR of DSPE-PEG 2000 -NHS (vehicle), DSPE-PEG 2000 -peptide and peptide (peptide: M27). b MALDI-TOF–MS of DSPE-PEG 2000 -peptide (peptide: M27). c Size, PDI, encapsulation efficiency, drug loading content and zeta-potential of nanovaccines. d Size of nanovaccines. e The transmission electron microscopy (TEM) image of nanovaccines. f Curves of peptide (M27) release from nanovaccines in different solutions. g Proportion of mature DC (CD11c + CD80 + CD86 + ) after incubation with normal saline (NS), peptide (Tyrp1, M20 or M27) or nanovaccines (Tyrp1-NP, M20-NP or M27-NP) for 48 h. P -values were determined by one-way ANOVA with Tukey’s multiple comparisons test. ** P = 0.0026 (M20 vs M20-NP), ** P = 0.0029 (NS vs M27), * P = 0.0429 (M27 vs M27-NP), *** P < 0.001

Article Snippet: Inguinal lymph nodes were obtained 48 h after immunization, made into frozen sections, and incubated with anti-CD3 rat monoclonal antibody (1:200) (Abcam, UK), anti-CD11c rabbit monoclonal antibody (1:200) (Cell Signaling Technology, USA) overnight at 4 °C.

Techniques: In Vitro, Activation Assay, Encapsulation, Zeta Potential Analyzer, Transmission Assay, Electron Microscopy, Incubation, Saline

Journal: Journal of Nanobiotechnology

Article Title: Lymph node-targeted neoantigen nanovaccines potentiate anti-tumor immune responses of post-surgical melanoma

doi: 10.1186/s12951-022-01397-7

Figure Lengend Snippet: Lymph node-targeting characteristic of neoantigen nanovaccines. a Equivalent peptide-NIR797 (Free vaccine group) and DSPE-PEG 2000 -peptide-NIR797 (Nanovaccine group) were separately mixed with Montanide™ ISA 51 and injected subcutaneously at the tail base of mice. The fluorescence distribution in mice at different time points was photographed by small animal in vivo imaging (n = 3). b Fluorescence image of inguinal lymph nodes 48 h after injection. c The average radiant efficiency of inguinal lymph nodes, spleens and kidneys 48 h after injection. P -values were determined by one-way ANOVA with Tukey’s multiple comparisons test. ** P = 0.0062. d – e A FITC labeled model antigen ovalbumin (OVA) was used to evaluate the distribution of nanovaccines. d Localization of nanovaccines and CD3 + T cells in inguinal lymph nodes 48 h after subcutaneous injection of DSPE-PEG 2000 -OVA-FITC, was shown by immunofluorescence staining. Nanovaccine: green (FITC); T cells (CD3): gray (Cy5); Scale: 500 μm. e Localization of nanovaccines and DCs in lymph nodes 48 h after subcutaneous injection of DSPE-PEG 2000 -OVA-FITC. Nanovaccine: green (FITC); DCs (CD11c): red (Cy3); Scale: 25 μm

Article Snippet: Inguinal lymph nodes were obtained 48 h after immunization, made into frozen sections, and incubated with anti-CD3 rat monoclonal antibody (1:200) (Abcam, UK), anti-CD11c rabbit monoclonal antibody (1:200) (Cell Signaling Technology, USA) overnight at 4 °C.

Techniques: Injection, Fluorescence, In Vivo Imaging, Labeling, Immunofluorescence, Staining

Journal: Journal of Nanobiotechnology

Article Title: Lymph node-targeted neoantigen nanovaccines potentiate anti-tumor immune responses of post-surgical melanoma

doi: 10.1186/s12951-022-01397-7

Figure Lengend Snippet: T cell responses activated by neoantigen nanovaccines. One week after last treatment, Proportions of mature DCs (CD11c + CD80 + CD86 + ) in lymph nodes ( a ), proportions of neoantigen specific T cells (CD3 + CD8 + M27-H 2 K b+ ) in spleens ( b ) and tumors ( c ), and proportions of effector memory T cells (CD3 + CD8 + CD44 + CD62L − ) in spleens ( d ) were analyzed by flow cytometry. P -values were determined by one-way ANOVA with Tukey’s multiple comparisons test. ** P = 0.0034 ( a ), ** P = 0.0026 (d, NS vs Nanovaccine), ** P = 0.0056 (d, Free vaccine vs Nanovaccine), *** P < 0.001. e Lymphocytes in spleens were incubated with CFSE labeled B16F10 melanoma cells and MFC forestomach cancer cells at effector-to-target ratio (E: T) of 10:1. PI was added 4 h after incubation and the percentage of dead tumor cells (CFSE + PI + / CFSE + ) was analyzed by flow cytometry. P -values were determined by one-way ANOVA with Tukey’s multiple comparisons test. * P = 0.0114 (NS vs Free vaccine), ** P = 0.0046 (Free vaccine vs Nanovaccine). f Cytokines in the supernatant after co-incubation of lymphocytes and tumor cells. P -values were determined by two-way ANOVA with Tukey’s HSD multiple comparison post hoc test. *** P < 0.001. The level of TNF-α ( g ) and IL-6 ( h ) in the tumor microenvironment. i Proportions of regulatory T cells (CD3 + CD4 + Foxp3 + ) in the tumor microenvironment. j The expression of PD-L1 in tumors. P -values were determined by one-way ANOVA with Tukey’s multiple comparisons test. ** P = 0.0014 ( g ), *** P < 0.001

Article Snippet: Inguinal lymph nodes were obtained 48 h after immunization, made into frozen sections, and incubated with anti-CD3 rat monoclonal antibody (1:200) (Abcam, UK), anti-CD11c rabbit monoclonal antibody (1:200) (Cell Signaling Technology, USA) overnight at 4 °C.

Techniques: Flow Cytometry, Incubation, Labeling, Comparison, Expressing

Journal: Cell Death & Disease

Article Title: ALCAM regulates multiple myeloma chemoresistant side population

doi: 10.1038/s41419-022-04556-8

Figure Lengend Snippet: A Hoechst staining of RPMI8226 cells in the presence of an EGF neutralizing antibody (10 μg/ml) for 24 h and the result quantification (right panel). B Hoechst staining of CTR-KD or AL-KD RPMI8226 cells in the presence of recombinant EGF protein (10 ng/ml) for 24 h and the result quantification (right panel). C Hoechst staining of AL-KD1 and AL-KD2 RPMI8226 cells, cocultured with BMSC in the presence of ALCAM-Fc chimera fusion protein (AL-Fc, 0.5 μg/ml) for 24 h and the result quantification (right panel). D Hoechst staining of CTR-KD or AL-KD RPMI8226 cells, cocultured with BMSC in the presence of EGFR inhibitor (gefitinib, 200 nΜ) for 24 h and the result quantification (right panel). E Hoechst staining of RPMI8226 cells in the presence of CD6 antibody (10 μg/ml) and the result quantification (right panel). Data are the mean of three independent experiments in three replicates. * p < 0.05.

Article Snippet: Anti-EGF (#AF236) and anti-CD6 (#AF627) neutralizing antibodies were ordered from R&D Systems Inc. EGFR inhibitor gefitinib (#S1025), SMO inhibitor cyclopamine (#S1146), and Gli1 inhibitor GANT61 (#S8075) were ordered from Selleck Inc. Two mitochondrial respiratory chain inhibitors—oligomycin (#C3007) and rotenone (#B5462)—were ordered from ApexBio Technology.

Techniques: Staining, Recombinant

Journal: Cell Death & Disease

Article Title: ALCAM regulates multiple myeloma chemoresistant side population

doi: 10.1038/s41419-022-04556-8

Figure Lengend Snippet: A MM cells RPMI8226, either CTR-KD or AL-KD, were treated with melphalan (Mel, 15 μM) or bortezomib (BTZ, 5 nM) for 24 h. The SP cell ratio was examined by Hoechst staining. B The RPMI8226 cells were treated by melphalan as described above. The cell cycle was analyzed after Hoechst staining. C Cell-cycle quantification. D After Hoechst staining, the apoptotic cells were analyzed by annexin V staining. E ALCAM and EGFR expression on MM cells after Mel or BTZ treatment were detected by flow cytometry. MFI mean fluorescence index. F Examination of SP cells after EGFR inhibitor (gefitinib, 200 nM) and melphalan treatment. Data are the mean of three independent experiments in three replicates. * p < 0.05.

Article Snippet: Anti-EGF (#AF236) and anti-CD6 (#AF627) neutralizing antibodies were ordered from R&D Systems Inc. EGFR inhibitor gefitinib (#S1025), SMO inhibitor cyclopamine (#S1146), and Gli1 inhibitor GANT61 (#S8075) were ordered from Selleck Inc. Two mitochondrial respiratory chain inhibitors—oligomycin (#C3007) and rotenone (#B5462)—were ordered from ApexBio Technology.

Techniques: Staining, Expressing, Flow Cytometry, Fluorescence

Journal: Cell Death & Disease

Article Title: ALCAM regulates multiple myeloma chemoresistant side population

doi: 10.1038/s41419-022-04556-8

Figure Lengend Snippet: A Scheme graph showing the animal study to evaluate the efficacy of the combination therapy (Mel and EGFR inhibitor) in vivo. The mice were treated by intraperitoneal injection of melphalan (60 μg/mouse per time, 4 times within 10 days) or gefitinib (500 μg/mouse per time, 4 times within 10 days), or a combination of both. Each group contained 8 mice. B Tumor-bearing mice were subjected to in vivo bioluminescent imaging (IVIS) before and after treatment. Five out of seven representative results are shown. C The relative luciferase activity of IVIS was calculated. D The tumor-bearing mice were treated twice as described above. Then, mice BM cells were analyzed by Hoechst staining for MM SP in vivo. Two out of three representative results are shown in the left panel, and result quantification is shown in the right panel. E Tumor burden was evaluated by circulating IgG2b. F Treatment efficacy was evaluated by mouse survival. G MM tumor-bearing mice were treated by intraperitoneal injection of PBS, bortezomib (15 μg/mouse per time, 4 times within 10 days), or a combination of bortezomib and gefitinib (500 μg/mouse per time, 4 times within 10 days). The PBS group contained 4 mice; the other treated group each contained 6 mice. Tumor burden was evaluated by circulating IgG2b. H Treatment efficacy was evaluated by mouse survival (* p < 0.05).

Article Snippet: Anti-EGF (#AF236) and anti-CD6 (#AF627) neutralizing antibodies were ordered from R&D Systems Inc. EGFR inhibitor gefitinib (#S1025), SMO inhibitor cyclopamine (#S1146), and Gli1 inhibitor GANT61 (#S8075) were ordered from Selleck Inc. Two mitochondrial respiratory chain inhibitors—oligomycin (#C3007) and rotenone (#B5462)—were ordered from ApexBio Technology.

Techniques: In Vivo, Injection, Imaging, Luciferase, Activity Assay, Staining

Journal: Cell Death & Disease

Article Title: ALCAM regulates multiple myeloma chemoresistant side population

doi: 10.1038/s41419-022-04556-8

Figure Lengend Snippet: A ALDEFLUOR analysis of BM cells from non-tumorous donors ( n = 5) and MM patients ( n = 7). Two representative results are shown. B BM cells from 42 newly diagnosed MM patients were analyzed by flow cytometry for ALCAM expression in CD138 + MM cells and ALDEFLUOR staining. Results show 2 representatives of ALCAM-high MM versus ALCAM-low MM, and corresponding ALDEFLUOR staining results. C Graphic model of EGFR-targeting therapy in MM treatment (* p < 0.05).

Article Snippet: Anti-EGF (#AF236) and anti-CD6 (#AF627) neutralizing antibodies were ordered from R&D Systems Inc. EGFR inhibitor gefitinib (#S1025), SMO inhibitor cyclopamine (#S1146), and Gli1 inhibitor GANT61 (#S8075) were ordered from Selleck Inc. Two mitochondrial respiratory chain inhibitors—oligomycin (#C3007) and rotenone (#B5462)—were ordered from ApexBio Technology.

Techniques: Flow Cytometry, Expressing, Staining

Journal: Molecular Therapy Oncolytics

Article Title: Nanobody-based retargeting of an oncolytic herpesvirus for eliminating CXCR4 + GBM cells: A proof of principle

doi: 10.1016/j.omto.2022.06.002

Figure Lengend Snippet: Efficacy of the oHSV detargeting and retargeting (A) Detargeting was evaluated by infection of J1.1–2, J/A (J1.1 HVEM + ), and J/C (J1.1 nectin-1 + ) cells infected for 72 h at different MOIs with the recombinant oHSVs expressing either wild-type (WT) gD (oHSV/gD) or gD modified by the insertion of an anti-hCXCR4 nanobody (oHSV/Nb-gD). Both viruses express EGFP under the control of pICP6, allowing the visualization of infected cells by epifluorescence microscopy. Scale bars represent 5 mm. (B and C) Retargeting was evaluated on U87MG (B) and U87MG CXCR4 + (C) cells. Cells were plated in 96-well plates, infected with oHSV/gD or oHSV/Nb-gD (MOI 0.1) and incubated in Incucyte S3 for real-time analyses during 72 h. EGFP expression and cell confluency were quantified every 6 h. Circles represent the ratio between the green and the phase area expressed as the mean ± SEM of four wells. Statistical significance was determined by ordinary two-way ANOVA with Bonferroni multiple comparisons of means with a single pooled variance (ns, non-significant; ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗∗ p < 0.0001). Images were taken every 6 h, and representative images taken at 72 hpi are shown. Scale bars represent 2 mm. Additional representative whole-well images taken at 24, 48, and 72 h are shown in Figure S2 . See also growth curve of oHSV/gD and oHSV/Nb-gD in U87MG CXCR4 + cells in Figure S3

Article Snippet: Vero cells (ATCC; no. CCL-81) and human glioblastoma U87MG (ATCC; no. HTB-14) cells were maintained in Dulbecco’s modified Eagle medium (DMEM) (Biowest, VWR Inernational, Leuven, Belgium) supplemented with 10% fetal bovine serum (FBS).

Techniques: Infection, Recombinant, Expressing, Modification, Control, Epifluorescence Microscopy, Incubation

Journal: Molecular Therapy Oncolytics

Article Title: Nanobody-based retargeting of an oncolytic herpesvirus for eliminating CXCR4 + GBM cells: A proof of principle

doi: 10.1016/j.omto.2022.06.002

Figure Lengend Snippet: Efficacy of the oHSV retargeting in patient-derived GSCs (A) Patient-derived GSCs (T08, T013, T018, and T033), U87MG, or U87MG CXCR4 + cells were cultured as tumorospheres and further dissociated for flow cytometry quantification of the percentage of cells expressing CXCR4 (APC + ) at the cell membrane. Bars represent the means ± SEM of four independent experiments. Statistical significance was determined by Kruskal-Wallis (primary cells, ∗∗p < 0.01) or Mann-Whitney (U87MG cells, ∗p < 0.05) test. (B) Overlayed histograms of a representative analysis allowing the comparison between endogenous and ectopic CXCR4 expression. Stemness features (expression of SOX2, POUF3, and SALL2) analyzed by qRT-PCR are depicted in . (C) Tumorospheres cultured in 24-well plates were infected with oHSV/gD or oHSV/Nb-gD (10 6 PFUs/mL). Forty-eight hours post-infection, cells were dissociated and the EGFP fluorescence was analyzed by flow cytometry. Bars represent the means ± SEM of three independent experiments. Statistical significance was determined by ordinary two-way ANOVA with Bonferroni’s multiple comparisons of means (∗∗p < 0.01). Raw data (overlaid histograms) representative of one experiment are shown in . (D) Tumorospheres cultures in 24-well plates and infected for 48 h by oHSV/Nb-gD (10 6 PFUs/mL) were either analyzed by epifluorescence for EGFP detection (left panels) or fixed for immunostaining of nestin (white) or CXCR4 (red) and GFP detection (green). Nuclei were labeled with DAPI (blue). Images were recorded with a NIKON A1R confocal microscope. Scale bars represent 100 μm. See also for real-time EGFP quantification and images of T033 tumorospheres infected with oHSV/gD or oHSV/Nb-gD at a higher titer (10 7 PFUs/mL).

Article Snippet: Vero cells (ATCC; no. CCL-81) and human glioblastoma U87MG (ATCC; no. HTB-14) cells were maintained in Dulbecco’s modified Eagle medium (DMEM) (Biowest, VWR Inernational, Leuven, Belgium) supplemented with 10% fetal bovine serum (FBS).

Techniques: Derivative Assay, Cell Culture, Flow Cytometry, Expressing, Membrane, MANN-WHITNEY, Comparison, Quantitative RT-PCR, Infection, Fluorescence, Immunostaining, Labeling, Microscopy

Journal: Molecular Therapy Oncolytics

Article Title: Nanobody-based retargeting of an oncolytic herpesvirus for eliminating CXCR4 + GBM cells: A proof of principle

doi: 10.1016/j.omto.2022.06.002

Figure Lengend Snippet: Efficacy of the oHSV arming (A) The replication efficacy of the non-armed (oHSV/Nb-gD) and sTRAIL-armed (oHSV/Nb-gD:sTRAIL) oncolytic viruses was evaluated with a growth curve assay. U87MG CXCR4 + cells were infected at a MOI of 1, and supernatant was harvested 24, 48, and 72 h post-infection and used for titration as previously described. The number of foci was calculated based on Incucyte S3 imaging. Bars represent the means ± SEM (PFUs/mL) of three independent experiments. The lack of statistical difference is confirmed by unpaired t test analysis. (B) PARP and caspase 3 cleavage was evaluated by western blot analysis on total cell extracts from U87MG CXCR4 + cells infected for 18 h by oHSV/Nb-gD or oHSV/Nb-gD:sTRAIL (MOI: 0.5 or 1). gD and α-tubulin detection were used as infection or loading control, respectively. (C) Apoptosis was measured at different time points by flow cytometry using annexin V/DAPI labeling of U87MG CXCR4 + cells infected by oHSV/Nb-gD or oHSV/Nb-gD:sTRAIL (MOI: 5). The percentage of apoptotic cells corresponds to early (annexin V + /DAPI − ) and late apoptotic (annexin V + /DAPI + ) cells. Percentages of apoptotic cells upon infection at other MOI (1, 5, and 10) are shown in . In parallel, cells were incubated with resazurin to evaluate the viability upon oHSV infection. Bars (percentage of apoptotic cells) and dots (percentage of viability) represent the means ± SEM of three independent experiments. Statistical significance was determined by ordinary two-way ANOVA with Bonferroni’s multiple comparisons of means (∗∗∗p < 0.001). (D and E) Patient-derived GSCs (T08, T013, T018, and T033) were cultured as tumorospheres in 24-well plates and infected with oHSV/Nb-gD or oHSV/Nb-gD:sTRAIL (10 6 PFUs/mL). gD and sTRAIL relative expression was analyzed 48 hpi by qRT-PCR as illustrated by a representative experiment. gD (D) and sTRAIL (E) mRNA level in oHSV/Nb-gD:sTRAIL-infected T08 are considered as the baseline (ND, not detected).

Article Snippet: Vero cells (ATCC; no. CCL-81) and human glioblastoma U87MG (ATCC; no. HTB-14) cells were maintained in Dulbecco’s modified Eagle medium (DMEM) (Biowest, VWR Inernational, Leuven, Belgium) supplemented with 10% fetal bovine serum (FBS).

Techniques: Infection, Titration, Imaging, Western Blot, Control, Flow Cytometry, Labeling, Incubation, Derivative Assay, Cell Culture, Expressing, Quantitative RT-PCR

Journal: Molecular Therapy Oncolytics

Article Title: Nanobody-based retargeting of an oncolytic herpesvirus for eliminating CXCR4 + GBM cells: A proof of principle

doi: 10.1016/j.omto.2022.06.002

Figure Lengend Snippet: In vivo efficacy of oHSV/Nb-gD and oHSV/Nb-gD:sTRAIL (A) Schematic representation of the experimental settings. Nude mice were engrafted with U87MG CXCR4 + Luc + cells and virus or PBS was injected in the tumor on day 7. Mice were sacrificed on day 22 (n = 9 in each group). (B) Mice were regularly weighed, and for each mouse, the weight change is expressed as a percentage to the weight on day 0, considered as equal to 100%. (C) Bioluminescence activity was recorded with Xenogen IVIS 50 on day 6, 13, and 20 after engraftment. See also Figure S9 for bioluminescence imaging. (B) and (C) represent the means ± SEM (n = 9 in each group). Statistical significance was determined by two-way ANOVA with Tukey’s multiple comparisons of means (∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001). (D–F) On day 22, brain from five mice were sectioned for immunostaining of human vimentin and the measurement of the tumor volume by 3D reconstruction (D) Data represent the means ± SEM. Statistical significance was determined by Kruskal-Wallis test (∗p < 0.05). Representative pictures of serial sections of two mice/group as well as the estimated volume of the corresponding tumor are shown in (E). In parallel, brain from the four other mice were divided into three parts (frontal, middle, and occipital), which were frozen and treated independently for RNA extraction and qRT-PCR analysis of hCXCR4 expression (F). For each sample, PBS-treated mice (middle sample) are considered as the baseline. Bars represent the means ± SEM. Statistical significance was determined by two-way ANOVA with Tukey’s multiple comparisons of means with a single pooled variance (∗p < 0.05; ∗∗p < 0.01).

Article Snippet: Vero cells (ATCC; no. CCL-81) and human glioblastoma U87MG (ATCC; no. HTB-14) cells were maintained in Dulbecco’s modified Eagle medium (DMEM) (Biowest, VWR Inernational, Leuven, Belgium) supplemented with 10% fetal bovine serum (FBS).

Techniques: In Vivo, Virus, Injection, Activity Assay, Imaging, Immunostaining, RNA Extraction, Quantitative RT-PCR, Expressing

Journal: Molecular Therapy Oncolytics

Article Title: Nanobody-based retargeting of an oncolytic herpesvirus for eliminating CXCR4 + GBM cells: A proof of principle

doi: 10.1016/j.omto.2022.06.002

Figure Lengend Snippet: Survival assay upon oHSV/Nb-gD or oHSV/Nb-gD:sTRAIL treatment (A) Schematic representation of the survival assay experimental settings. (B) Bioluminescence activity of nude mice engrafted with 5 × 10 4 U87MG CXCR4 Luc + cells was recorded with Xenogen IVIS 50 on day 5 (2 days before treatment) and 13 (6 days after treatment). Bars represent the means ± SEM. Statistical significance was determined by two-way ANOVA with Tukey’s multiple comparisons of means. (∗∗p < 0.01). See also Figure S10 B for bioluminescence imaging. (C) Probability of survival of mice treated with PBS (n = 7), oHSV/Nb-gD (n = 6), or oHSV/Nb-gD:sTRAIL (n = 5). The red arrow indicates the day of treatment (day 7). Statistical significance was determined by log rank (Mantel-Cox) test (∗∗∗∗p < 0.0001). See also Figure S10 A for weight follow-up.

Article Snippet: Vero cells (ATCC; no. CCL-81) and human glioblastoma U87MG (ATCC; no. HTB-14) cells were maintained in Dulbecco’s modified Eagle medium (DMEM) (Biowest, VWR Inernational, Leuven, Belgium) supplemented with 10% fetal bovine serum (FBS).

Techniques: Clonogenic Cell Survival Assay, Activity Assay, Imaging