Journal: Acta Neuropathologica

Article Title: Neuroinflammatory disease signatures in SPG11 -related hereditary spastic paraplegia patients

doi: 10.1007/s00401-023-02675-w

Figure Lengend Snippet: Ruxolitinib inhibits SPG11 iMGL hyperactivation and rescues toxicity induced in SPG11 neurons. a Representative immunofluorescence of control and SPG11 iMGL (IFNγ: 10 ng/µl for 24 h, Ruxolitinib: 50 µM for 24 h before IFNγ treatment) stained for IBA1, STAT1 and CXCL10. Scale bar 50 µm. b Quantification of mean fluorescence intensity of STAT1 and CXCL10 within IBA1 + control ( n = 4) and SPG11 ( n = 4) iMGL. Each dot represents the mean of cells within five random fields of view (0.15 mm 2 ). Bars represent means ± SD. P value according to two-way ANOVA with Bonferroni’s multiple comparison test . c Schematic representation of the experimental paradigm. Control and SPG11 iMGL were either treated with Ruxolitinib and IFNγ, with IFNγ only or remained untreated. Microglia-conditioned media (MCM) was collected, diluted 1:1 with neuronal media and added to control and SPG11 neurons that were differentiated for 2 weeks from NPCs. After 48 h, cell death was measured by ICC. d Representative images of TUBB3 and DAPI staining in SPG11 neurons treated with MCM derived from non-treated (NT), IFNγ treated, and Ruxolitinib (Rux) and IFNγ treated SPG11 iMGL. Apoptotic cells were visualized by TUNEL assay. Scale bar: 50 µm. e Quantification of apoptotic cells by TUNEL signal intensity within the DAPI + nuclei of TUBB3 + control ( n = 1) and SPG11 ( n = 1) neurons treated with MCM derived from control ( n = 5) and SPG11 ( n = 5) iMGL. Each dot represents the mean of cells within five random fields of view (0.15 mm 2 ). Bars represent means ± SD. P value according to one-way ANOVA with Bonferroni’s multiple comparison test . ns P > 0.05; * P < 0.05; ** P < 0.01; NT non-treated; Rux Ruxolitinib; MCM microglia-conditioned media

Article Snippet: For visualization of apoptotic cells, the One-step TUNEL In Situ Apoptosis Kit (Red, Elab Fluor® 647; Elabscience) was used before antibody staining according to the manufacturer’s instructions.

Techniques: Immunofluorescence, Control, Staining, Fluorescence, Comparison, Derivative Assay, TUNEL Assay

Journal: PLOS One

Article Title: Inhibition of HER2 signaling and breast cancer cell growth with a novel antibody targeting HER2 ECD III/IV

doi: 10.1371/journal.pone.0338127

Figure Lengend Snippet: A. Competitive ELISA assessing the binding of His-tagged IgGs to immobilized HER2 in the presence or absence of saturating concentrations of non-His-tagged IgGs, detected with an anti-His-HRP antibody. B. Saturation binding curves of trastuzumab and pertuzumab to HER2 on live MCF7 cells. Cells were incubated with antibodies (0-400 nM) for 30 min and detected with an AF647-conjugated goat anti-human secondary antibody via flow cytometry. C. Competitive binding of of AF647-labeled trastuzumab or pertuzumab (0-300 nM) to MCF7 cells pre-saturated with 267 nM of unlabeled trastuzumab and pertuzumab, analyzed by flow cytometry. D. Binding of AF647-conjugated m66 or m75 to MCF7 cells after pre-incubation with 267 nM trastuzumab or pertuzumab. E. Binding of AF647-conjugated rabbit antibodies to MCF7 cells pre-saturated with 267 nM of trastuzumab and pertuzumab, evaluated by flow cytometry. T, trastuzumab; P, pertuzumab. Error bars, SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001, T-test.

Article Snippet: Briefly, each antibody was reacted with 20 molar equivalents of AF647 NHS ester (Biotium, 30–3007) for 2 h at room temperature protected from light.

Techniques: Competitive ELISA, Binding Assay, Incubation, Flow Cytometry, Labeling