Journal: Materials Today Bio

Article Title: Silk films with distinct surface topography modulate plasma membrane curvature to polarize macrophages

doi: 10.1016/j.mtbio.2024.101193

Figure Lengend Snippet: Potential mechanism of surface roughness-induced macrophage polarization. (a) Western blot images of αv integrin expression in PS and YS film-treated RAW 264.7 cells for 24 h. (b) Immunofluorescent images of cellular distribution αv integrin in PS and YS film-treated RAW 264.7 cells. Scale bar in low magnification, 10 μm. Scale bar in high magnification, 5 μm. (c) Mean optical intensity of αv integrin from (b). (d) Western blot images of the expressions of FAK, phosphorylated-FAK, NF-κB, and phosphorylated–NF–κB in cells grown on PS and YS films. (e, f) Quantitative analysis of p-FAK and p–NF–κB in (d). (g) Thin-section transmission electron microscope (TEM) images of PS and YS films. Scale bar, 1 μm. (h,i) Thin-section TEM images of RAW 264.7 cell-PS/YS film interface. Cells were highlighted in red. Scale bar in (h), 5 μm. Scale bar in (i), 200 nm. (j,k) Co-localization of αv integrin and lysosomes in cells on PS and YS film-treated cells for 24 h. Scale bar, 10 μm. Lysosomes were stained by the lysotracker red dye. (l) Possible mechanism of the effect of surface roughness on macrophage polarization. Data are expressed as mean ± SD (n = 3). * p < 0.05, ** p < 0.01, two-tailed student's t-test. PS: as cast slow drying film, YS: salting out-treated slow drying film.

Article Snippet: Inc. ELISA kits (TNF-α, IL-6, IL-10, TGF-β) were purchased from Elabscience, Inc. Integrin Alpha V Polyclonal antibody (27096-1-AP) was purchased from Proteintech Group, Inc. PE-conjugated anti-mouse CD206 (141705) and PE-conjugated anti-mouse CD80 (50-201-4699) antibodies were purchased from Biolegend, Inc, and Tonbo Biosciences, Inc, respectively.

Techniques: Western Blot, Expressing, Transmission Assay, Microscopy, Staining, Two Tailed Test, Salting Out

Journal: Advanced Science

Article Title: Calpain‐2‐Mediated Endothelial Focal Adhesion Disruption in Thoracic Aortic Dissection

doi: 10.1002/advs.202501112

Figure Lengend Snippet: Conditional knockout of capns1 in endothelial cells prevented the formation of BAPN‐induced TAAD. A) En‐face immunofluorescence staining images depicting Calpain‐1, Calpain‐2, Capns1, and endothelial marker VE‐cadherin (VEC) in the descending aorta from Capns1 F/F and Capns1 △EC mice. Nuclei were counter‐stained using DAPI. Scale bar, 10 µm. B) The survival rate was estimated using the Kaplan‐Meier method and compared via the log‐rank test. C) The incidence of TAAD in Capns1 F/F and Capns1 △EC mice following 4 weeks of BAPN treatment. Saline‐Capns1 F/F , n = 6; Saline‐Capns1 △EC , n = 8; BAPN‐Capns1 F/F , n = 20; BAPN‐Capns1 △EC , n = 22. D) Quantification of the maximal diameters of both the ascending and descending aorta over the 4‐week period post‐BAPN treatment. Statistical significances were determined by two‐way ANOVA with Sidak's multiple comparision. E) Gross images depict the morphology of the entire aorta. Scale bar: 5 mm. F) The permeation of Evans blue dye into the thoracic aortas of Capns1 F/F and Capns1 △EC mice as assessed at 7 and 14 days after BAPN administration. G) Quantitative data on mRNA expression levels for Itgav, Vcl, and TIn1 ( n = 3 per group). Statistical significances were determined by two‐way ANOVA with Tukey's multiple comparision. H) Hematoxylin and eosin(HE), elastic van Gieson (EVG), and immunofluorescence (IF) staining of Itgav and Vinculin in cross‐sections of the Capns1 F/F and Capns1 △EC aorta.

Article Snippet: Antibodies for α‐SMA (rabbit IgG, 14395‐1‐AP), CD31 (rabbit IgG, 11265‐1‐AP), CD68 (rabbit IgG, 28058‐1‐AP), MMP9 (rabbit IgG, 10375‐2‐AP), Calpain‐2 (rabbit IgG, 11472‐1‐AP), Calpain‐1 (rabbit IgG, 10538‐1‐AP), VE‐cadherin (mouse IgG, 66804‐1‐Ig), Itgav (rabbit IgG, 27096‐1‐AP), Itgb3 (rabbit IgG, 18309‐1‐AP), Talin‐1 (rabbit IgG, 14168‐1‐AP), and Vinculin (mouse IgG, 66305‐1‐Ig) were sourced from Proteintech, China.

Techniques: Knock-Out, Immunofluorescence, Staining, Marker, Saline, Expressing

Journal: Advanced Science

Article Title: Calpain‐2‐Mediated Endothelial Focal Adhesion Disruption in Thoracic Aortic Dissection

doi: 10.1002/advs.202501112

Figure Lengend Snippet: The regulation of integrin activation and talin cleavage by Calpain‐2 in endothelial cells. Human aortic endothelial cells (HAECs) were transduced with Calpain‐2 via lentiviral transfection and subsequently treated with 1 µmol Angiotensin II (Ang II) for 24 h. A) The expression levels of Calpain‐2, Itgav, Itgb3, Talin, Vinculin, and VE‐cadherin were assessed using Western blotting. Representative Western blot images were presented, with GAPDH serving as the loading control. B) Quantitative data from the Western blot analysis ( n = 3 per group). C) Representative images of immunofluorescence staining for Itgav and VE‐cadherin in HAECs. Scale bar: 10 µm. D) Cell lysates from HAECs underwent co‐immunoprecipitation (Co‐IP) using anti‐Calpain‐2 or anti‐Itgav antibodies, followed by Western blotting. E) HA‐Capn2 or Flag‐Itgav were co‐transfected into HEK293T cells, demonstrating the colocalization of Calpain‐2 (green) and Itgav (red) in these cells. Scale bar:10 um. F) An adhesion assay was performed to evaluate the adhesion of THP‐1 monocytes to HAECs. Representative images showing DiI‐labeled THP‐1 monocyte adhesion to HAECs. Scale bar: 20 um. G) The relative number of THP‐1 monocytes adhering to HAECs following exposure to 1 µ m Ang II for 24 h was presented. Statistical significances were determined by One‐way ANOVA with Tukey's multiple comparision.

Article Snippet: Antibodies for α‐SMA (rabbit IgG, 14395‐1‐AP), CD31 (rabbit IgG, 11265‐1‐AP), CD68 (rabbit IgG, 28058‐1‐AP), MMP9 (rabbit IgG, 10375‐2‐AP), Calpain‐2 (rabbit IgG, 11472‐1‐AP), Calpain‐1 (rabbit IgG, 10538‐1‐AP), VE‐cadherin (mouse IgG, 66804‐1‐Ig), Itgav (rabbit IgG, 27096‐1‐AP), Itgb3 (rabbit IgG, 18309‐1‐AP), Talin‐1 (rabbit IgG, 14168‐1‐AP), and Vinculin (mouse IgG, 66305‐1‐Ig) were sourced from Proteintech, China.

Techniques: Activation Assay, Transduction, Transfection, Expressing, Western Blot, Control, Immunofluorescence, Staining, Immunoprecipitation, Co-Immunoprecipitation Assay, Cell Adhesion Assay, Labeling

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Self-Triggered Apoptosis Enzyme Prodrug Therapy (STAEPT): Enhancing Targeted Therapies via Recurrent Bystander Killing Effect by Exploiting Caspase-Cleavable Linker.

doi: 10.1002/advs.201800368

Figure Lengend Snippet: Figure 2. Enhanced cellular uptake and cytotoxic activity of RGDEVD-DOX in integrin αvβ3 expressing cells. a) Representative confocal images (left) and quantitative analysis (right) of HDMEC and U-87 MG cells exposed to fluorescent-labeled RDEVD and RGDEVD peptides. b) Representative confocal images (left) and quantitative analysis (right) of scrambled control and integrin αv (ITGAV) siRNA-transfected HDMECs and U-87 MG cells exposed to fluorescent-labeled RGDEVD peptide. Green and blue indicate the fluorescent-labeled peptides and cell nuclei, respectively. Scale bar, 50 µm. c) Flow cytometry analysis of isotype control (top), scrambled control-transfected (lower left), and ITGAV siRNA-transfected (lower right) U87 MG cells incubated with fluorescent-labeled RGDEVD and stained with an antibody against integrin αvβ3. d) Representative confocal images (left) and quantitative analysis (right) of U-87 MG and HT-29 cells treated with RDEVD-DOX and RGDEVD-DOX. Red and blue indicate the intrinsic red fluorescence of doxorubicin and cell nuclei, respectively. Scale bar, 50 µm. e) Concentration-dependent cytotoxicity of RDEVD-DOX and RGDEVD-DOX on U-87 MG (left) and HT-29 (right) determined by MTT assay (n = 4). f) Doxorubicin release from RGDEVD-DOX when incubated in PBS (pH 7.4) containing (or not containing) carboxylesterase (n = 3). Data are mean ± s.d. *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: No peak corresponding to Cy5.5-maleimide was detected, indicating the complete removal of unreacted dye. siRNA Transfection: Transient gene knockdown of HDMEC and U-87 MG was carried out using siRNA duplex against ITGAV (ITGAV Trilencer-27 human siRNA; OriGene, Rockville, MD; cat. no. SR302468) or a scrambled negative control siRNA (OriGene; Cat. No. SR30004) following the manufacturer’s instruction.

Techniques: Activity Assay, Expressing, Labeling, Control, Transfection, Flow Cytometry, Incubation, Staining, Fluorescence, Concentration Assay, MTT Assay