Journal: Cell Death & Disease

Article Title: Fibrillar α-synuclein induces neurotoxic astrocyte activation via RIP kinase signaling and NF-κB

doi: 10.1038/s41419-021-04049-0

Figure Lengend Snippet: a–d qRT-PCR analysis of indicated genes in human midbrain astrocyte cultures treated following 24 h PFF treatment ± cotreatment with JSH-23. e Confocal microscopy of NF-κB component p65 (red) and nuclei (DAPI, blue) in human midbrain astrocytes following 2 h PFF treatment ± cotreatment with BAY. Scale bars = 20 μm. f Colocalization of red and blue signal in ( e ) is quantified as Fisher’s Z transformed index of correlation. g Nuclear fractions were isolated from human midbrain astrocyte cultures following 2 h PFF treatment ± cotreatment with BAY. Relative levels of p65 in nuclear fractions were quantified using ELISA. h , i Secondary analysis of microarray profiling of ( h ) rat astrocytes treated with conditioned neuronal medium containing LacZ or α-synuclein (GSE11574) or i human postmortem substantia nigra samples from patients with PD or healthy controls (GSE26927). Z scores were calculated for genes under the GO term 0038061 and those exhibiting significantly differential expression between groups (corrected p value < 0.05) are displayed in the respective heatmaps. j–m qRT-PCR analysis of indicated genes in human midbrain astrocyte cultures treated following 24 h PFF treatment ± cotreatment with indicated inhibitors. ns not significant, * p < 0.05, ** p < 0.01, *** p < 0.001. Bars represent group means. n = 6 independent replicates for all experiments.

Article Snippet: Equal amounts of protein were then processed through an ELISA-based kit for detecting p65 (NF-κB p65 Transcription Factor Assay Kit, Abcam, Cambridge, MA, #ab133112).

Techniques: Quantitative RT-PCR, Confocal Microscopy, Transformation Assay, Isolation, Enzyme-linked Immunosorbent Assay, Microarray, Quantitative Proteomics

Journal: Cell Death & Disease

Article Title: Fibrillar α-synuclein induces neurotoxic astrocyte activation via RIP kinase signaling and NF-κB

doi: 10.1038/s41419-021-04049-0

Figure Lengend Snippet: a Confocal microscopy of NF-κB component p65 (red) and nuclei (DAPI, blue) in human midbrain astrocytes following 2 h PFF treatment ± cotreatment with indicated inhibitors. Scale bars = 50 μm. b Colocalization of red and blue signal in ( a ) is quantified as Fisher’s Z transformed index of correlation. c Nuclear fractions were isolated from human midbrain astrocyte cultures following 2 h PFF treatment ± cotreatment with indicated inhibitors. Relative levels of p65 in nuclear fractions were quantified using ELISA. d–j Primary human midbrain astrocyte cultures were treated with PFFs or PBS control solution. Cultures were pretreated (30 min) with inhibitors of RIPK3 (GSK872) or NF-κB (BAY) signaling prior to the addition of PFFs. Levels of indicated transcripts were measured at indicated time points using qRT-PCR. ns not significant, * p < 0.05, ** p < 0.01, *** p < 0.001. Bars represent group means. n = 6 independent replicates in ( a–c ). n = 3 independent replicates in ( d–j ).

Article Snippet: Equal amounts of protein were then processed through an ELISA-based kit for detecting p65 (NF-κB p65 Transcription Factor Assay Kit, Abcam, Cambridge, MA, #ab133112).

Techniques: Confocal Microscopy, Transformation Assay, Isolation, Enzyme-linked Immunosorbent Assay, Control, Quantitative RT-PCR

Journal: Life Science Alliance

Article Title: HB-EGF activates EGFR to induce reactive neural stem cells in the mouse hippocampus after seizures

doi: 10.26508/lsa.202201840

Figure Lengend Snippet: HB-EGF increases early after the induction of MTLE in vivo and is secreted by NSPCs in vitro. (A) Confocal microscopy images from Nestin-GFP mice, 14dpKA showing the reduction caused by gefitinib of the MTLE-induced GCD and NSC overactivation. Scale bar, 20 μm. (B) Quantification of the volume of the GCL in saline+DMSO–, saline+ gefitinib–, KA+DMSO–, and KA+gefitinib–treated animals 14dpKA. (C) Quantification of the BrdU+ cells in saline+DMSO–, saline+ gefitinib–, KA+DMSO–, and KA+gefitinib–treated animals 14dpKA. (D) Quantification of BrdU+ NSCs, ANPs, and overall cells in the SGZ expressed as the percentage with respect to KA+DMSO animals. (E) Confocal microscopy images showing an increase in the EGFR ligand HB-EGF in the hilus and the molecular layer of Nestin-GFP mice 3dpKA. Scale bar, 20 μm. (F) Quantification of HB-EGF by ELISA at different time points during the first 3dpKA. Values are represented as the pg of HB-EGF in 100 μg of the tissue. (G) Immunofluorescence images of NSPCs in vitro showing HB-EGF expression. Scale bar, 10 μm. (H) Quantification by ELISA of HB-EGF released in vitro, expressed as the pg of the ligand per mL. Dots show individual data. Data information: for (B), Kruskal–Wallis test, * P < 0.05, * P < 0.01, and *** P < 0.001. Bars show the mean ± SEM. Dots show individual data. For (C), Kruskal–Wallis test, * P < 0.05, * P < 0.01, and *** P < 0.001. Bars show the mean ± SEM. Dots show individual data. For (D), t test, ** P < 0.01. Bars show the mean ± SEM. Dots show individual data. For (F), one-way ANOVA, * P < 0.05 and *** P < 0.001. Bars show the mean ± SEM. Dots show individual data. For (H), Kruskal–Wallis test, * P < 0.05. Bars show the mean ± SEM. Source data are available for this figure.

Article Snippet: Next, we determined the amount of HB-EGF per cell through ELISA (Cat#DY8239-05; R&D Systems).

Techniques: In Vivo, In Vitro, Confocal Microscopy, Saline, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Expressing

Journal: Journal for Immunotherapy of Cancer

Article Title: TSH-TSHR axis promotes tumor immune evasion

doi: 10.1136/jitc-2021-004049

Figure Lengend Snippet: Expression and release of TSH in moDCs (A–D) Analysis of single-cell RNA sequencing data of thyroid tissues integrated from five patients with anaplastic thyroid cancer and two healthy persons. (A–B) UMAP plot of all cells from thyroid tissues, which visualize cell expression profiles in a two-dimensional independent space. Cells are colored based on clusters defined by cell type (A) and TSHα expression (B). (C–D) TSHα expression in different cell populations (C) and myeloid cells subpopulations (D). (E) Expression of TSHα and TSHβ2 in different immune cells through qRT-PCR assay. (F) Whole-cell lysates from immature and mature moDCs were subjected to immunoblot analysis of TSHα and TSHβ2. (G) Expression of TSHα and TSHβ2 in moDCs from healthy donors and patients with DTC through quantitative RT-PCR assay. (H) Secretion of TSH in moDCs from healthy donors and patients with DTC through ELISA assay. (I) Immunofluorescence staining of myeloid cells for CD11c (red), TSHα (white), and TSHβ (green). Representative images of three independent experiments with similar results are shown. *P<0.05, **p<0.01, ***p<0.001. DTC, differentiated thyroid cancer; moDCs, monocyte-derived dendritic cells; cDC, classic DC; pDC, plasmacytoid DC; TSH, thyroid-stimulating hormone; TPM, transcript per million.

Article Snippet: Then TSH concentration in the cell-free supernatants was measured through Human Thyroid Stimulating Hormone ELISA Kit (Abcam) following the kit protocol.

Techniques: Expressing, RNA Sequencing Assay, Quantitative RT-PCR, Western Blot, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Staining, Derivative Assay