Journal: STAR Protocols

Article Title: Immunofluorescence staining of phosphoinositides in primary mouse hippocampal neurons in dissociated culture

doi: 10.1016/j.xpro.2022.101549

Figure Lengend Snippet:

Article Snippet: Incubate neurons with primary antibodies (PI3P, Z-P003, Echelon, 1:50, PI4P, Z-P004, Echelon, 1:200, or PI(4,5)P 2 , Z-A045, Echelon, 1:150 and Rab8, R5530, Sigma-Aldrich, 1:150 or TGN46, ab50595, Abcam, 1:200) in buffer A containing 5% NGS for 1 h. 41.

Techniques: Recombinant, Electron Microscopy, Software, Microscopy, Cell Culture, In Vitro, Cell Counting

Journal: Oncogenesis

Article Title: Regulation of cargo transfer between ESCRT-0 and ESCRT-I complexes by flotillin-1 during endosomal sorting of ubiquitinated cargo

doi: 10.1038/oncsis.2017.47

Figure Lengend Snippet: EGFR degradation is impaired in the absence of flotillin-1.( a , b ) Control siRNA, flotillin-1 (F1-siRNAa and -b) and ( c , d ) control HeLa or flotillin-1-knockout cells (transfected with flotillin-1-EGFP or not) were starved overnight and stimulated with 100 ng/ml EGF for 60 min, fixed with methanol and immunostained for EGFR. ( b ) The amount of undegraded EGFR in flotillin-1-knockdown cells was quantified as fluorescence intensity in arbitrary units using the ImageJ Software. A significant increase of EGFR signal was observed in flotillin-knockdown cells as compared with the control. ( d ) EGFR amount after 60 min was quantified as in c , and the data show a rescue of EGFR degradation upon flotillin-1-EGFP expression (cells marked with *, images for GFP in ). The data in c , d are shown as mean±s.d. Cells from three independent experiments were evaluated (control siRNA cells: n =136, F1-siRNAa: n =135 and F1-siRNAb: n =149; control HeLa: n =358, flotillin-1 KO: n =181, rescue cells: n =252). Statistical analysis was performed with one-way analysis of variance (ANOVA) and Bonferroni post-test in comparison with the control. *** P <0.001. Scale bar in ( a , c , e ): 10 μm. ( e ) Flotillin-1-knockdown cells (F1-siRNAb) were treated as in a and immunostained for EGFR (green) and Rab5, Hrs or LAMP1 (red).

Article Snippet: Mouse monoclonal antibody against Hrs (sc-271455, clone C-7; WB: 1:1000), CD63/LAMP3 (sc-5275; IF: 1:200), ubiquitin (sc-8017, clone P4D1; WB: 1:1000, IF: 1:100) and EGFR (sc-120, clone 528; IF: 1:100, WB: 1:1000) as well as rabbit polyclonal antibodies against Hrs (sc-30221, clone M-79; IF: 1:40, WB: 1:1000; IP: 1:100), Rab5 (sc-46692; IF: 1:200) and c-myc (sc-789; IP: 1:300) were from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Control, Knock-Out, Transfection, Knockdown, Fluorescence, Software, Expressing, Comparison

Journal: Oncogenesis

Article Title: Regulation of cargo transfer between ESCRT-0 and ESCRT-I complexes by flotillin-1 during endosomal sorting of ubiquitinated cargo

doi: 10.1038/oncsis.2017.47

Figure Lengend Snippet: Endosomal morphology is altered in flotillin-1-knockdown/knockout cells, but endosomal domains persist.( a ) Control and flotillin-1-knockout cells were chased for 2 h with Fe-EGF, fixed and analysed by transmission electron microscopy. Note the enlarged endosomes in flotillin-1-knockdown cells. Scale bar: 500 nm. ( b ) Control siRNA and flotillin-1-knockdown cells were stimulated with EGF for 30 min and labelled with an anti-mouse LAMP3 antibody detected with a secondary Alexa Fluor 647-coupled antibody. The specimens were analysed with Leica SR GSD 3D microscope. Scale bar: 2 μm. ( c ) Rab5-Q79L-GFP-expressing HeLa cells were immunostained for endogenous flotillin-2 and Hrs, which show a limited colocalization in the enlarged endosomes. Scale bar: 10 μm. ( d ) Rab5-Q79L-GFP-expressing HeLa and flotillin-1-knockout cells were immunostained for endogenous CHC and Hrs, which are localised in endosomal domains in both cell types and show some colocalization. Scale bar: 10 μm.

Article Snippet: Mouse monoclonal antibody against Hrs (sc-271455, clone C-7; WB: 1:1000), CD63/LAMP3 (sc-5275; IF: 1:200), ubiquitin (sc-8017, clone P4D1; WB: 1:1000, IF: 1:100) and EGFR (sc-120, clone 528; IF: 1:100, WB: 1:1000) as well as rabbit polyclonal antibodies against Hrs (sc-30221, clone M-79; IF: 1:40, WB: 1:1000; IP: 1:100), Rab5 (sc-46692; IF: 1:200) and c-myc (sc-789; IP: 1:300) were from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Knockdown, Knock-Out, Control, Transmission Assay, Electron Microscopy, Microscopy, Expressing

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: Nature Communications

Article Title: Flexopiezoelectricity at ferroelastic domain walls in WO 3 films

doi: 10.1038/s41467-020-18644-w

Figure Lengend Snippet: a The surface morphology of a 610-nm-thick film with four-oriented monoclinic unit cells. The white dashed lines represent the macro-domain walls. The nearby crooked lines are step edges with a single unit-cell height indicating the films were grown in the step-flow mode. b The cross-sectional TEM (dark field) image taken with reflection g = \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$[{\mathrm{00}}\bar 2]_{\mathrm{WO}}$$\end{document} [ 00 2 ¯ ] WO shows the alternating A and B domains with fine- and macro-domain walls in a ~300-nm-thick film. c, d Zoomed-in bright-field images along the [001] YAO ( c ) and \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$[{\mathrm{1}}\bar 1{\mathrm{0}}]$$\end{document} [ 1 1 ¯ 0 ] YAO ( d ) zone axes. e, f SAED patterns of the A and B domains, taken along the \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$[{\mathrm{1}}\bar 1{\mathrm{0}}]$$\end{document} [ 1 1 ¯ 0 ] YAO zone axis. A white (or red) box represents the pseudocubic unit cell (or monoclinic unit cell with eight octahedrons). The monoclinic unit cells in real space are shown and the subscript ‘ WO ’ represents the crystal axes in the monoclinic cell. From a crystallographic viewpoint, we categorize the macro-domains into A or B domains, depending on whether the [010] WO axis (with lattice constant b ) of the monoclinic unit cell is parallel to the substrate \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$[{\mathrm{1}}\bar 1{\mathrm{0}}]$$\end{document} [ 1 1 ¯ 0 ] YAO or \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$[{\mathrm{001}}]$$\end{document} [ 001 ] YAO . Scale bars indicate 200 nm.

Article Snippet: Cross-sectional bright-field (BF) and dark-field (DF) TEM imaging were performed with a 200 kV field-emission TEM (JEM-2100F, JEOL) equipped with a spherical aberration corrector (CEOS GmbH).

Techniques: