Journal: Journal of hazardous materials

Article Title: Mechanistic insight into airborne particulate matter PM10 as an environmental hazard for hemorrhagic stroke: Evidence from in vitro and in vivo studies.

doi: 10.1016/j.jhazmat.2024.136319

Figure Lengend Snippet: Fig. 5. PM10 promotes CA development through FasL-mediated necroptosis of VSMC in the cerebrovasculature. Representative immunofluorescence staining images of (a) p-RIPK1 and (b) FasL in 3D vascular spheroids following PM10 exposure. All sections were counterstained with anti-α −SMA antibody for VSMC as well as DAPI for nuclei (x400 magnification). Histograms represent the quantification of fluorescence intensity. The fluorescence intensity profile of FasL expression is shown. (c) Immunofluorescence staining for NF-κB p65 nuclear translocation in the vascular cell culture spheroid model system in response to PM10 (x400 magnification). The fluorescence intensity profile of the p65 localization is shown. (d and e) Assessment of the efficacy of the superoxide-dependent Fenton reaction in the vascular spheroids upon PM10 exposure, evaluated via fluorescent probes corresponding to intracellular labile iron pools (Calcein-AM) and superoxide radicals (HKSOX-1). (f) Immunohistochemical staining for p-MLKL in the internal carotid artery (ICA) (x400 magnification). (g) Immunofluorescence for p-RIPK3 with green fluorescence in ICA, counterstained with α-SMA (red) and DAPI (blue) (x400 magnification). (h) Immunofluorescence staining for NF-κB p65 nuclear translocation in the ICA following intratracheal PM10 instillation in mice (x400 magnification). The fluorescence intensity profile of the p65 localization is shown. (i) Representative immunofluorescence images for FasL upregulation in the ICA in response to PM10 exposure. (j) Serological levels of S100B, a diagnostic marker for hemorrhagic stroke, were assessed by ELISA in serum. (k) Immunofluorescence staining for myeloperoxidase (MPO) in the ICA, indicating increased circulating neutrophil infiltration into the cerebral vasculature following PM10 inhalation (x400 magnification). All data are presented as mean ± standard error of the mean, and *in dicates statistically significant differences between groups at *p < 0.05 (n = 6).

Article Snippet: HITB5 (CLU305), vascular smooth muscle cell (VSMC), was purchased from CEDARLANE (ON, Canada).

Techniques: Immunofluorescence, Staining, Fluorescence, Expressing, Translocation Assay, Cell Culture, Immunohistochemical staining, Diagnostic Assay, Marker, Enzyme-linked Immunosorbent Assay

Journal: Nature Communications

Article Title: Cell shape information is transduced through tension-independent mechanisms

doi: 10.1038/s41467-017-02218-4

Figure Lengend Snippet: Shape signals control maturation of vascular smooth muscle cells. a Representative images of SMCs plated on ellipsoid micropatterns and stained either for (left) α-SMA (green) and F-actin (red), or (right) calponin (green) and F-actin (red). In untreated SMCs, both α-SMA and calponin showed increased expression with increasing aspect ratio that was colocalized with actin stress fibers, which is a hallmark of contractile SMC maturation. When integrin β 3 activation was blocked, this phenotypic feature was abolished. Treated cells with β 1 blocking antibodies had no effect on the shape-driven phenotype even though the cells failed to comply with the ellipsoid micropatterns. b Quantitative analysis of α-SMA and calponin in SMCs plated on ellipsoid patterns with or without integrin β 1 or β 3 blocking antibodies. Values are given as mean ± SEM; n = 80, chosen randomly from eight different slides cultured independently (^ p < 0.05, * p < 0.01 vs. previous ratio; one-way ANOVA comparisons independent for each condition). c SMCs were treated with varying concentrations of blebbistatin from 0.1 to 100 μM for 12 h before fixation and stained for F-actin (red) and α-SMA (green). Both stress fiber integrity and compliance of the cells with the micropatterns decrease with increasing blebbistatin concentration; however, α-SMA expression shows little change with the increasing blebbistatin concentration

Article Snippet: HITB5 human vascular smooth muscle cells from Cellutions Biosystems were cultured in M199 medium (25 mM HEPES, Invitrogen, Cat: 12340–030) supplemented with 10% fetal bovine serum, and 100 units/ml penicillin, and maintained in 5% CO 2 at 37 °C.

Techniques: Control, Staining, Expressing, Activation Assay, Blocking Assay, Cell Culture, Concentration Assay