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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 <t>TUNEL</t> 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
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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 <t>TUNEL</t> 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
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an alexa fluor 647 anti-human foxp3 staining kit  (Becton Dickinson)
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Becton Dickinson an alexa fluor 647 anti-human foxp3 staining kit
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 <t>TUNEL</t> 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
An Alexa Fluor 647 Anti Human Foxp3 Staining Kit, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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hilyte fluor 647 labeling kit  (Dojindo Labs)
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Dojindo Labs hilyte fluor 647 labeling kit
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 <t>TUNEL</t> 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
Hilyte Fluor 647 Labeling Kit, supplied by Dojindo Labs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Thermo Fisher alexa fluor 647 tyramide superboost kit streptavidin
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 <t>TUNEL</t> 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
Alexa Fluor 647 Tyramide Superboost Kit Streptavidin, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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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 <t>TUNEL</t> 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
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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 <t>TUNEL</t> 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
Lightning Link Rapid Alexa Fluor 647, supplied by Novus Biologicals, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Thermo Fisher ares alexa fluor 647 dna labeling kit
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 <t>TUNEL</t> 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
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Image Search Results


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

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